A reliable and sensitive method for the determination of pyronaridine in human blood was developed and validated. A 0.3 ml aliquot of whole blood was extracted using liquid-liquid extraction after addition of amodiaquine as an internal standard. Analysis was performed on a Shimadzu LCMS-2010A in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Separation was achieved on a Gemini 5 microm C18 3.0 x 150 mm column using a mobile phase composed of 2mM perflurooctanoic acid-acetonitrile mixture delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.2 and 8.2 min, respectively, with a total run time of 14 min. The estimated calibration range of the method was 5.7-855 ng/mL. The analysis of quality control samples for pyronaridine at 11.4, 285, and 760 ng/mL demonstrated excellent precision with relative standard deviation of 11.1, 4.8 and 2.2%, respectively (n=5). Recoveries at concentrations of 11.4, 285 and 760 ng/mL were all greater than 75%. No interference peaks or matrix effects were observed. This LC-MS method for the determination of pyronaridine in human blood has excellent specifications for sensitivity, reproducibility and accuracy. This LC-MS technique was found to improve the quantitation of pyronaridine in whole blood allowing its use in pharmacokinetic studies with clinically relevant doses.