The topic of attribution and authentication of art works is a well-known one and is currently the subject of heated debate. To arrive at a univocal scientific truth, however, it is necessary to integrate historical-humanistic and technical-experimental skills with a subjective and objective evaluation. The first part of the volume deals with experimentation relating to three valuable artifacts of different material composition: a painting, a sculpture, a codex. The second part of the research regards the emblematic case of the Mona Lisa and its uniqueness. The question connected to this study is: "Is there a second Mona Lisa?" Is the Isleworth Mona Lisa, also known as the Earlier Mona Lisa, a second version of the Louvre Mona Lisa painted by the Renaissance genius Leonardo da Vinci? In order to provide an answer regarding attribution, a methodological path was applied to the painting on canvas "Mona Lisa with columns" (St. Petersburg), establishing that it was a copy, albeit a well-executed one. Numerous versions and copies of the Mona Lisa were also studied, basing their examination on the in-depth archival-bibliographic research of the many paintings on the same subject. The findings highlighted the distinction between two authentic versions by Leonardo, i.e. the Louvre Mona Lisa and the Isleworth Mona Lisa and two copies, i.e. the Prado and the Reynolds Mona Lisas. The latter two, considered to be the most complete and qualitatively better that many others are, in fact, either copies of previous Mona Lisas or of the two authentic versions. The final considerations are dedicated to the chronological sequence of historical sources, stylistic and aesthetic analyses, artistic techniques and experimental investigations to prove the authentication of the Isleworth Mona Lisa by Leonardo.