Ocrelizumab is a monoclonal anti-CD20 antibody approved for the treatment of multiple sclerosis (MS). The clinical value of therapeutic drug monitoring (TDM) for this antibody in treatment of MS is unknown, and an adequately specific and precise quantitation method for ocrelizumab in patient serum could facilitate investigation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitation methods have been shown to have higher analytic specificity and precision than enzyme-linked immunosorbent assays. To establish and validate an LC-MS/MS-based quantitation method for ocrelizumab. We present an LC-MS/MS-based quantitation method using immunocapture purification followed by trypsinization and analysis by a triple quadrupole mass analyzer obtaining results within the same day. We found that the ocrelizumab peptide GLEWVGAIYPGNGDTSYNQK (Q1/Q3 Quantifier ion: 723.683+/590.77 y112+ Qualifier ion: 723.683+/672.30 y122+) can be used for quantitation and thereby developed a method for quantifying ocrelizumab in human serum with a quantitation range of 1.56 to 200 µg/mL. The method was validated in accordance with EMA requirements in terms of selectivity, carry-over, lower limit of quantitation, calibration curve, accuracy, precision and matrix effect. Ocrelizumab serum concentrations were measured in three MS patients treated with ocrelizumab, immediately before and after ocrelizumab infusion, with additional sampling after 2, 4, 8 and 12 weeks. Measured serum concentrations of ocrelizumab showed expected values for both Cmax and drug half-life over the sampled time period. We have established a reliable quantitation method for serum ocrelizumab that can be applied in clinical studies, facilitating the evaluation of ocrelizumab TDM in MS.