A bioanalytical method was developed and validated for the quantification of capreomycin (Cm) analogs, Cm IA and Cm IB, in human plasma. This implemented ion-pairing solid phase extraction, followed by ion-pairing high-performance liquid chromatography, with tandem mass spectrometry detection. Chromatographic separation was achieved using a Discovery C18 , 5 μm, 4.6 × 50 mm analytical column. An isocratic mobile phase consisting of water and acetonitrile with 0.1% formic acid and 4mm heptafluorobutyric acid (80:20; v/v) was used at a flow-rate of 500 μL/min. An AB Sciex API 3000 mass spectrometer at unit resolution, in multiple reaction monitoring mode, was used for detection. Electrospray ionization was used for ion production. The method was successfully validated for the range 469-30,000 ng/mL for Cm IA and for Cm IB, with cefotaxime as the internal standard. The within- and between-day precision determinations for Cm IA and IB, expressed as the percentage coefficient of variation, were < 20.0% at the lower limit of quantification (LLOQ) and < 8.2% at all other test concentrations. Recovery of both analogs was > 72.3% and reproducible at the low, medium and high end of the calibration range. No significant matrix effects were observed for the analyte. The assay performed well when applied to clinical samples generated from children in a clinical multidrug resistant tuberculosis research study in South Africa.