STV said tonight it is going ahead with plans to screen the head to head between the First Minister and the leader of the No to independence campaign on August 5.
Glasgow 2014 officially ends on August 3.
The two politicians will square up during the two hour programme, which will be broadcast live from 8pm to 10pm at The Royal Conservatoire of Scotland in Glasgow.
The announcement follows months of wrangling between the Yes Scotland campaign and Better Together over when and where it would take place.
STV had originally suggested July 16, with an Edinburgh broadcast, between the two figureheads. But this was rejected by Mr Salmond, although it was accepted by the former Labour Chancellor. The SNP leader wanted to only go up against Prime Minister David Cameron on that date.
But the broadcaster mooted August 5 after Mr Salmond said he would debate Mr Darling only after the Commonwealth Games is over. This decision led to criticism of STV by Better Together, who said it had bowed to pressure from the Nationalists.
There was also talk of a BBC televised debate on August 12, from Inverness.
A spokesman for the First Minister said: "We accepted this invitation weeks ago and are delighted that Alistair Darling has finally done likewise and accepted the challenge of debating with the First Minister on live TV, in front of a large audience of voters.
"Mr Darling will be acting as a shield for the Prime Minister - who we will continue to pursue for a debate - and as such he will be defending the Tory policies of David Cameron's government.
"The choice in this referendum is between continued austerity with Westminster or new prosperity and a fairer country in an independent Scotland - that is the message we will take into this debate and which will win the day in September."
The broadcaster's political editor Bernard Ponsonby will host the event in front of an audience of 350 members of the public that fairly represent views from all sides.
Gordon Macmillan, head of news at STV, said: "STV has demonstrated its commitment to providing a platform for debate on Scotland's constitutional future and securing this landmark TV debate on Tuesday 5th August confirms the next stage of our commitment. The live TV debate will give members of the public an opportunity to put questions to Alistair Darling and Alex Salmond and will be available to STV viewers and throughout the UK online."
Following the debate, a special edition of Scotland Tonight will feature highlights and analysis.
A spokesman for Better Together said: "We are delighted that there will be a live, televised debate between the leaders of both campaigns. Alex Salmond will now finally have to answer questions on the pound, pensions and public services that he has spent the last two years dodging."