At the start of the marathon session, Supreme Court President Ricardo Lewandowski reminded senators that they were about to "exercise one of the most serious tasks under the constitution."
The Senate president, Renan Calheiros, also underlined the gravity of the impeachment process, which Rousseff, a one-time Marxist guerrilla, has likened to a coup d'etat.
"I want to emphasize the gravity of the decision that we will soon take," he said. "I ask that we set aside as much as possible our party political convictions."
It is the final vote before the one that will decide Rousseff's ultimate political fate, when a two-thirds majority would be needed to strip her of her power and end 13 years of leftist rule in Brazil. That vote is expected to take place around the end of August, just days after the Rio Olympics end.
The Senate suspended Rousseff, Brazil's first woman president, on May 12 over accusations of illegal accounting practices and fiddling the budget to mask the slump in the economy.
Instead, unpopular interim president Michel Temer -- formerly the vice president -- is struggling to drag the country out of its worst recession in decades as the Senate debates what to do with his former boss and bitter enemy.
Rousseff is accused of spending money without congressional approval and taking out unauthorized loans from state banks to make the national budget look better than it really was as she campaigned for re-election in 2014.
Her allies from the Workers' Party point out that many of the lawmakers accusing her are implicated in corruption cases arguably far more serious than accounting tricks.
