A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-D -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150 × 4.6 mm; 3 μm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103-75 m/z for cycloserine and 124 to 80 m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3-30 μg/mL was established for cycloserine using 0.2 mL human plasma and a 1 μL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8-3.4 %; accuracy, 93.8-104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.