As a lieutenant to his cousin, the condottiero Gian Giacomo Medici (known as Medeghino), in the fall of 1531, Serbelloni fought against the Sforza militias and the imperial forces of Charles V, who later became his lord and employer.

In 1551, still with 300 men on foot, Serbelloni was sent by Ferrante Gonzaga to defend Asti against the French; he conquered the town of Saluzzo and was appointed as its governor.

After serving as overseer of the fortresses for Duke Cosimo II de' Medici, Serbelloni's career was advanced when another of his cousins was elected Pope as Pius IV.

Serbelloni entered the ranks of Knights of Malta, sending troops to help break the Turkish siege and organizing naval raids against the Muslims.

In 1567, Serbelloni followed the Spanish armies into Belgium under the Duke of Alva during the repression of the Dutch Revolt, being appointed twice as governor of Antwerp.

The following year Ottoman forces under the command of Sinan Pasha besieged and eventually took Tunis; Serbelloni was humiliated, dragged by his beard to the conqueror to ask for mercy on his knees, and many of his surviving men were slain.