The Battle of Pitreavie, on July 20, 1651, marked the end of Scotland's last hold-out against subjugation by Oliver Cromwell's New Model Army.
It is thought the bodies of 2000 Scots soldiers are buried on the site, which is currently agricultural land.
An archaeological evaluation is being prepared for the site at Rosyth, near Dunfermline, after Fife Council won consent to turn it into a rail, bus and car interchange.
It is now seeking tenders for archaeological works which will test for the presence of human remains.
The clash at Pitreavie was the final part of the Battle of Inverkeithing, the bloody end of Scotland's defence against the invading English army.
Historic Scotland's inventory of battlefields describes Inverkeithing as the last major battle of the Wars of the Three Kingdoms in Scotland, with Scotland falling under the control of the Protectorate from 1652.
On July 17, 1651, a 1600-strong Cromwellian force of cavalry and infantry began an assault on the Scottish cannon battery on the top of the Ferryhills, which had stopped Cromwell crossing the Forth.
"Cromwell had been trying to cross the Forth since his victory at Dunbar almost a year earlier but he knew that attempting a crossing in the teeth of the cannon battery would have been suicidal," said Fife Council archaeologist, Douglas Speirs.
The English forces successfully stormed the batteries, encouraging Cromwell to quickly send over another 4000 troops.
A Scottish force of some 5500 engaged them but it was quickly routed. Speirs added: "They retreated some four kilometres before regrouping on open ground just to the south of Pitreavie Castle but they were no match for Cromwell's vastly superior professional New Model Army and in less than two hours, the Scottish army was all but annihilated.
"There is no single contemporary record that can be relied upon for an accurate body count but from piecing various bits of historical information together it appears likely that between 1500 and 2000 Scots soldiers died.
"The battle at Pitreavie, which witnessed a great loss of life, was an incredibly important national event which, although little remembered in the national consciousness, deserves to be up there with Culloden, Bannockburn and Pinkie.
"It was truly a seminal battle in defining the future and the political landscape of Scotland, defining every aspect of Scotland from 1651 until the Restoration in 1660."
The battlefield site is largely uncommemorated although a small cairn was erected a few years ago to mark the 500 members of the clan Maclean who perished at Cromwell's hands.
It is likely there are a number of mass burial pits, although only two have ever been found, during ditch-digging in the 1850s.
The area has been developed in the past so a test excavation will sample it to see whether any battlefield deposits remain.
"The results will inform any more detailed archaeological work that may be required to preserve the site by record before any development," said Speirs.
It is likely musket balls, cannon shot and smaller personal items such as buttons or plaid brooches will be uncovered in the process.