A selective and sensitive analytical method for the simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC–MS was developed. After liquid–liquid extraction with dichlormethane, separation was achieved on a Phenomenex Luna Pentafluorophenyl Column (150 mm × 2 mm, 5 μm) using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis Q1 Scans with negative ionisation were chosen. The method was validated for serum as the matrix of choice. Limits of detection are in the picogram range, limits of quantification are between 0.05 and 0.42 ng/mL, recoveries are above 50%. The assay was linear in the calibration range from 1.0 to 50 ng/mL for cucurbitacin E and from 0.10 to 50 ng/mL for the cucurbitacins B, I and E-glucoside. The applicability of the method was demonstrated by the determination of cucurbitacins in zucchini plant material and body fluids from intoxication cases.