Artemether–lumefantrine is the most widely recommended artemisinin-based combination treatment for falciparum malaria. Quantification of artemether and its metabolite dihydroartemisinin in biological matrices has traditionally been difficult, with sensitivity being an issue. A high-throughput bioanalytical method for the analysis of artemether and its metabolite dihydroartemisinin in human plasma using solid-phase extraction in the 96-well plate format and liquid chromatography coupled to positive ion mode tandem mass spectroscopy has been developed and validated according to US FDA guidelines. The method uses 50 µl plasma and covers the calibration range 1.43–500 ng/ml with a limit of detection at 0.36 ng/ml. The developed liquid chromatography–tandem mass spectrometry assay is more sensitive than all previous methods despite using a lower plasma volume (50 µl) and is highly suitable for clinical studies where plasma volumes are limited, such as pediatric trials.