A sensitive and specific method was developed and validated for the quantitation of maytansinoid (DM1) in human serum using on-line solid phase extraction (SPE)—liquid chromatography–tandem mass spectrometry (LC–MS/MS). Because DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum, samples were pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). The resulting samples were diluted with acetonitrile prior to the on-line solid phase extraction (SPE) on a C18 cartridge. A C18 (150 × 4.6 mm ID 3 μm particle size) column was used for chromatographic separation with a 10.0 min HPLC gradient and DM1-NEM was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. DM1 concentrations were back-calculated from DM1-NEM amount found in the human serum samples. The quantitation range of the method was 0.200–200 ng/mL when using 0.25 mL serum. Within-run day precisions (n = 6) were 0.9–4.4% and between-run day (3 days runs; n = 18) precisions 2.5–5.6%. Method biases were between 3.5–14.5% across the whole calibration range. DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. Finally, the assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload.