Abstract A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100μL), cells (2.5×105), or cell culture media (100μL) by LLE and separated on a Prodigy C18 (150mm×4.0mm, 5μm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5mL/min, with umbelliferone (600ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05–20μg/mL) and in vitro cells (0.78–50ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.