Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article describes the development and validation of a bioanalytical assay to quantify plitidepsin in human plasma, urine and whole blood using HPLC–MS/MS. The analyte was extracted from the matrix by liquid–liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column, gradient elution was applied for chromatographic separation and detection was performed on a triple quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range 0.1–100 ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of quantification of 0.1 ng/mL in all three matrices, allowing the quantification of trace levels of plitidepsin, and accomplishing this in an analysis time of two minutes only. The presented method was successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.