The results presented in this work show that: i) there is a direct, high-affinity interaction of plitidepsin with eEF1A2; ii) such interaction has also been observed in living tumor cells using a FLIM-phasor FRET approach; iii) in K-562 cancer cell lysates, both eEF1A isoforms were the only proteins that combined enough abundance and affinity to be detected as capable of binding plitidepsin; and iv) sensitivity to plitidepsin in the different cancer cell lines studied here is dependent on eEF1A2 levels. Here, EEF1A1 is linked to cancer.