A sensitive and high throughput bioanalytical method has been developed for reliable determination of amodiaquine (AQ), N-desethylamodiaquine (DEAQ), artesunate (AS) and dihydroartemisinin (DHA) in human plasma by LC–MS/MS. The method employs a solid phase extraction procedure without an evaporation step and with optimum use of organic solvents to circumvent degradation of artemisinin derivatives. The analytes and their deuterated internal standards (ISs) were analyzed on Hypersil Gold (100 mm × 4.6 mm, 5 μm) column using acetonitrile and 2.0 mM ammonium formate (pH 2.50) in 80:20 (v/v) ratio as the mobile phase. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was used to detect and quantify the analytes. The method was established over the concentration range of 0.250–30.0 ng/mL, 1.50–180 ng/mL, 2.00–600 ng/mL and 5.00–1400 ng/mL for AQ, DEAQ, AS and DHA respectively using 250 μL human plasma. The intra-day and inter-day accuracy and precision (% CV) across quality controls varied from 93.3–105.0% and 1.7–8.3 respectively for all the analytes. The stability was assessed in whole blood as well as in plasma samples under different conditions. All four analytes were stable in whole blood up to 2 h on melting ice. The long term stability in plasma was ascertained up to 90 days. IS-normalized matrix factors ranged from 0.988–1.023 for all the analytes. The method was successfully applied to a bioequivalence study using 50 mg artesunate and 135 mg amodiaquine fixed dose formulation in 14 healthy subjects.