SNP MP Joanna Cherry QC, English tax barrister Jo Maugham QC and businessman Dale Vince have launched a new action at the Court of Session, Scotland's highest civil court.
It also follows the passing of legislation, brought by Labour MP Hillary Benn, that makes it illegal for the United Kingdom government from pursuing a so called hard Brexit.
Mr Johnson will have to write to the European Union on 19 October to ask for an extra three months, unless he returns with a deal - then approved by MPs - or gets the Commons to back a no-deal Brexit.
But despite the new law, Mr Johnson said he would rather be "dead in a ditch" than ask for an extension.
Mid-October is when Johnson would have to come to Brussels for a summit of European Union leaders with either a new withdrawal agreement or a request for a delay to Brexit, but the British leader insists he would rather be "dead in a ditch" than postpone the October 31 withdrawal. "Er yes, indeed", said the prime minister.
He ruled that whether or not to prorogue Parliament was for politicians, not the courts, to decide.
Mr Johnson was not represented during the brief hearing.
But the Government is appealing and the case is scheduled to go to the Supreme Court on Tuesday.
Mr O'Neill added: "I could expand on why but I think it is self evident".
Johnson then talked over the protester saying he would be getting back to parliament "very soon".
The Prime Minister did not react as one man was heard telling him: "Find a deal here - this is Doncaster, not Europe".
"Whatever the shenanigans that may be going on at Westminster, we will get on with delivering our agenda and preparing to take this country out of the European Union on October 31", he said.
With less than 50 days until the United Kingdom is due to leave, the government and parliament are locked in conflict over the future of Brexit, with possible outcomes ranging from leaving without a deal to another referendum.
"Can you tell me why you are not with them in parliament sorting out the mess that you have created?" asked the heckler, to a low cheer from another part of the crowd.