David Cameron was heckled by Eurosceptics as he pledged to throw himself "headlong" into the campaign to keep Britain in the EU if his renegotiation of membership terms succeeds.
As the Prime Minister delivered a speech to the CBI conference in London, two protesters stood up and chanted "Voice of Brussels".
After telling them they were "making fools of yourselves", Mr Cameron insisted he had "no emotional attachment" to the institutions of the EU.
He said he wanted to "debunk" the idea that the UK could not survive outside the union.
"The argument is not whether Britain could survive outside the EU - the argument is how are we going to be better off," Mr Cameron said.
But the premier - who will set out his main reform demands in a letter to European Council president Donald Tusk on Tuesday - expressed hope that he and business leaders could throw themselves "headlong into keeping Britain in a reformed Europe" after a successful renegotiation.
As Mr Cameron spoke, two youths stood up holding a banner and began chanting: "CBI - voice of Brussels."
The PM said: "Come on, guys. If you sit down now, you can ask me a question rather than making fools of yourselves by just standing up and protesting."
The pair were quickly ushered out by security.
Mr Cameron said future links to the EU represented a "huge question for our country in the year ahead and until we have that referendum".
"I am not satisfied with the status quo we have got in Europe," he said. "The things I want fixed - whether it is making a more competitive Europe, whether it is making sure we are out of an ever-closer union, whether it is making sure there is proper fairness between those in the eurozone and those out of the eurozone ... or whether it is reducing the pressures that we face because of immigration - these are big and important changes and I think it is vital that we achieve them.
"Once I have achieved them, if I can achieve them, you will see me campaigning vigorously for Britain to stay in a reformed Europe.
"As I have said, if I cannot achieve them I rule nothing out."
Mr Cameron said he had been using the past few weeks to "debunk" arguments about the EU that he regarded as "duff".
He has already dismissed the idea that the UK would benefit from a Norway-style semi-detached relationship with the union, and said he was now tackling suggestions that the country could not survive outside the EU.
"When it comes to the crucial issues, our prosperity, our national security, of course we could try to look after those things outside the EU, but how do we make ourselves more prosperous and secure?" he said.
"We need to fix these challenges, fix these problems. That is what the renegotiation is about and then we can throw ourselves headlong into keeping Britain in a reformed Europe."
He added: "I don't have any emotional attachments to the institutions of the EU. But I have a very strong emotional and practical attachment to asking the simple questions for Britain - How do we have more influence in the world? How do we have more prosperity? How do we have more jobs? How do we do the best for this country that we love? To me that is what it is all about."
Mr Cameron said other EU leaders should be in no doubt that he was "deadly serious" about the UK's membership being seriously in question.
And he insisted he had not watered down demands for curbs on EU migrants' welfare claims.
"I couldn't have been more clear with my colleagues," he told the conference. "I have been to every single president and prime minister and I have very patiently set out what needs to change.
"But patiently setting out a list of very sensible changes should not be mistaken for a lack of resolve.
"If these things can't be fixed then Britain would naturally ask 'Do we belong in this organisation?'.
"In a way, you can boil down all of my negotiation to one word: flexibilty.
"Is this organisation flexible enough to make sure that countries inside the eurozone can grow and succeed and countries outside the eurozone, like Britain, can find what they need as well?
"If it's flexible enough, we'll stay. If it's not flexible enough, we will have to ask ourselves a very profound question: is this organisation for us?
"I think people in Europe know I am deadly serious about that and that is what the negotiation that we will be launching tomorrow is all about."
He went on: "On the migrant issue, we have not changed our view at all.
"We believe that what is set out in the manifesto is right for Britain, right for Europe and needs to change."
The protesters told Sky News outside the conference that they had been helped by the Vote Leave campaign group and were granted valid passes by the CBI.
"We got in because Vote Leave formed a company for us that was able to get us in. We managed to get our passes and we pretended to be businessmen," one said.
They described the experience as "terrifying" but insisted it had been "worth it" to highlight concerns.
"We were worried about the CBI misrepresenting British business's views ... in reality a lot of particularly small and medium sized businesses in Britain think the EU hinders rather than helps them," they added.