Grasso, an anti-mafia magistrate, was met with catcalls and cries of "you should be ashamed!" from the Forza Italia and other conservative senators.
"I am defending the dignity of the Senate," was Grasso's retort.
Berlusconi is facing trial this month for paying left-wing senator Sergio De Gregorio 3 million euros to defect to his conservative party in 2006 and help bring down the centre-left government of Romano Prodi.
Also charged in the latest case is Berlusconi's former aide, Valter Lavitola, who is accused of being an intermediary in the bribe.
Gregorio, who accused Berlusconi, was sentenced to 20 months in jail in an earlier plea bargain.
The 2008 collapse of the Prodi government triggered elections that returned Berlusconi to power for the third time.
The new trial is another legal setback for 77-year-old Berlusconi, who was banned from holding office for two years after his binding conviction for tax fraud in August. The billionaire media tycoon is facing a year's community service and was in November expelled from Italian Senate after his 1 August conviction by Italy's supreme court.