The original core of Palazzo Giustiniani was built at the end of the sixteenth century by Mgr Francesco Vento. The building was sold on 4 July 1590 to Giuseppe Giustiniani, a member of one of Genoa's richest and most powerful families. The Giustiniani family, at one time governors on behalf of Genoa of their native island of Chios, was forced to abandon the island in 1566 because of Turkish aggression.

They moved to Via della Dogana, in the financial and bureaucratic heart of papal Rome. In 1590 they bought the palazzo, which then took their name, and purchased other adjacent buildings over the following years. It would actually be more exact to talk in terms of merging various buildings, than the construction of a new palazzo, and the end result was a kind of "Giustiniani block" near the Pantheon. The family's art collection was housed in this building, which was rented out, fully or in part, to prelates and other eminent figures seeking prestigious accommodation.

In the course of later extension work the main façade overlooking Via della Dogana was completed, and the interior of the building was altered. Among the many frescoed rooms, mention should be made of the Zuccari Hall, named after the artist Federico Zuccari [1539-1609] who painted the fresco on the ceiling. This is the only room left untouched by the many alterations to the building. It is entirely decorated with frescoes: the ceiling depicts five episodes from the stories of Solomon [Solomon anointed king, the construction of the temple, the judgement of Solomon, the children forced to stab their father's corpse, and in the centre, Solomon meeting the Queen of Sheba] and the four virtues attributed to him [Religion, Industry, Vigilance and Eloquence].

Near the corners of the ceiling there are some small landscapes in round frames. The walls were graced with female figures representing the virtues, but today the only visible full figure still remaining is Temperance, although fragments of the others may also be seen. All the decorations are arranged on a surface painted with grotesques, creating a magnificent overall effect, which is further enhanced by seventeenth-century tapestries. The Giustiniani family line died out at the end of the nineteenth century, after which the huge art collection was broken up. But the Ministry of Education stepped in to halt this around the beginning of the twentieth century, transferring the most valuable pieces to the State.

At about that time, part of the property was taken over by the savings bank Cassa di Risparmio di Roma and another part was entrusted to the Grand Orient Masonic Lodge of Italy. At the beginning of 1926, Mussolini accepted a request from the then President of the Senate of the Kingdom, Tommaso Tittoni, to permit the Senate to use Palazzo Giustiniani, although the Senate did not acquire almost complete use of the building until 1988.

It was in this palazzo that Enrico De Nicola, then Italy's Acting Head of State, signed the Italian Constitution in 1947. The official apartment of the President of the Senate, the offices of ex officioand life senators and the Historical Archives, are currently located in the palazzo.