Malaria is one of the most important parasitic diseases of man. The development of drug resistance in malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has received renewed interest as a partner drug in artemisinin-based combination therapy. To study its pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a robust, highly sensitive and accurate bioanalytical method for drug measurements in biological samples. We have developed a sensitive quantification method that covers a wide range of clinically relevant concentrations (1.5 ng/mL to 882 ng/mL) using a relatively low volume sample of 100 μL of whole blood. Total run time is 5 min and precision is within ±15% at all concentration levels. Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a HALO RP amide fused-core column using a gradient mobile phase of acetonitrile–ammonium formate and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine accurately for up to 42 days after a single oral dose in a drug-drug interaction study in healthy volunteers.