Sensitive bioanalytical methods are required for pharmacokinetic studies in children, due to the small volume and modest number of samples that can be obtained. We sought to develop a LC–MS assay for primaquine and its active metabolite, carboxyprimaquine, following simultaneous, solid phase extraction of both analytes from human plasma. The analysis was conducted on a single-quad LC–MS system (Shimadzu Model 2020) in ESI+ mode, with quantitation by selected ion monitoring. Primaquine, carboxyprimaquine and 8-aminoquinoline (internal standard) were separated using a mobile phase of 80:20 methanol:water with 0.1% (v/v) formic acid and a Luna C 18 HPLC column, at ambient temperature. Solid phase extraction of the analytes from plasma (0.5mL) was achieved with Oasis ® HLB cartridges. The retention times for primaquine, 8-aminoquinoline and carboxyprimaquine were 3.3, 5.7 and 8.5min, respectively. The calibration curve range (2–1500μg/L) was appropriate for the limits of quantification and detection for primaquine (2μg/L and 1μg/L, respectively) and carboxyprimaquine (2.5μg/L and 1μg/L) and the anticipated plasma concentrations of the analytes. Intra- and inter-day precision for both primaquine and carboxyprimaquine was <10% across the concentration range 5–1000μg/L. Accuracy for both analytes was <15% (5–500μg/L). This validated LC–MS method with solid phase extraction facilitates the simultaneous analysis of primaquine and carboxyprimaquine from small volumes of human plasma, with run time <10min, recovery >85% and sensitivity of 1–2μg/L.