Judges in the Court of Appeal have refused a challenge from campaign groups, who now plan to take their fight to the Supreme Court.
Fifteen councils and other objectors had asked appeal judges to order further assessment of the scheme as a whole.
But Staffordshire businessman Trevor Forrester, who is hoping to mount the largest class action legal case in British history against HS2 Ltd and the Department for Transport, today vowed that the battle will continue despite yesterday's rejection.
"The rejection of the appeals is a blip, and now I think the class action case is the only way forward," he said.
"We know the business case for HS2 is flawed, and it has been a humiliating few weeks for HS2 Ltd with the costs escalating, the CBI withdrawing its support and Peter Mandelson expressing concerns. The fight will continue."
Mr Forrester is currently trying to generate support from 100,000 households along the route to back his fresh legal bid.
Meanwhile, resident and campaigner Rolfe Pearce, who lives in Colwich, near Rugeley, said: "A sad day comes to pass that people hurt by a government project are having to take the Government to court to plead for their environment and their lives and property.
"Far from losing this battle, we are now ever more emboldened that the Government's case is slowly slipping away from them, and support across the UK is slipping. So the campaign must ratchet up a gear for the appeal to the Supreme Court.
"This is now a fight I believe we can win. For the first time, I see the metal of the government corroding badly. They won the case just and as we gather ever more evidence we will show that the case for HS2 is not only flawed but now unsustainable."
Penny Gaines, chair of Stop HS2, said: "In the original ruling and the appeal, the judges have not looked at whether HS2 is a sensible way of spending £50billion of taxpayers' money. They've said it's up to Parliament to make the decision.
"But it is vital that Parliament has the right information they need on the environmental devastation HS2 will cause, and as yet, HS2 Ltd simply have not done the work."
The first phase of HS2 will stretch from London to Lichfield, with the second leg cutting through swathes of Staffordshire countryside stretching from Lichfield to Stone.
Phase one of the scheme is set to open in 2026, with the full Y-shaped second route open in 2032 to 2033.
But fears have been raised over the cost of the project, which currently stands at £42.6 billion, and the environmental impact it will have.
Fears have also been raised it will mean a reduction in the number of train services running in the Black Country and Staffordshire.
The original legal challenge brought by opponents to HS2 was thrown out in a ruling in March by Mr Justice Ouseley. Campaigners had to raise £100,000 to launch their appeal.