Tymoshenko spoke to a crowd estimated in the thousands at Independence Square, Kiev, the BBC reported, hours after she was released from prison -- where she had been held since 2011 after being convicted of abuse of power. Her appearance came one day after Parliament voted to set her free, and hours after the MPs voted to remove Yanukovych and call for early elections.
At least 88 people have been killed since Tuesday in violent protests in Kiev.
"You are heroes," Tymoshenko told fellow opposition members Saturday.
"Until you finish this job and until we travel all the way, nobody has the right to leave," she said. "Because nobody could do it -- not other countries, nobody -- could do what you have done. We've eliminated this cancer, this tumor."
The BBC said dozens of people walked away as Tymoshenko took the stage, shouting that she does not represent them.
The vote to remove Yanukovych came about an hour after said he would not resign.
Yanukovych said in a television interview from a city near the Russian border he would not leave office because he needed to "protect the people." He accused protesters of conducting a "coup."
The White House said Saturday the United States is "closely monitoring developments in Ukraine."
"We have consistently advocated a de-escalation of violence, constitutional change, a coalition government, and early elections, and today's developments could move us closer to that goal," White House press secretary Jay Carney said in a statement. "The unshakeable principle guiding events must be that the people of Ukraine determine their own future."
Carney said the United States welcomes Tymoshenko's release.
"Going forward, the Ukrainian people should know that the United States deeply values our longstanding ties with Ukraine and will support them as they pursue a path of democracy and economic development," he said.
Ukrainian lawmakers said early presidential elections would be held May 25. Opposition leaders had called for the early elections after a peace deal, struck Friday between the government and opposition leaders, stipulated elections would be held by December.
Speaker Volodymyr Rybak resigned when Parliament resumed Saturday and was replaced by Oleksandr Turchynov, an ally of Tymoshenko.
Police have abandoned their posts throughout Kiev, with the Interior Ministry issuing a statement that police were "at the service of the people and completely shares its aspirations for rapid changes."
The agreement mediated by the European Union calls for restoration of the 2004 constitution that limited the president's power. A unity government will be formed and constitutional reform begun to balance the powers of the various branches of government.