Purpose: Malaria is the most life-threatening endemic disease with high rate of morbidity and mortality. The WHO reported that 212 million new cases of malaria were identified globally in 2015, and children under age 5 are particularly susceptible to infection and death. A fixed-dose oral formulation of artemether (ARM) and lumefantrine (LFT) is recommended currently by WHO as the first line treatment for uncomplicated malaria. ARM is a semi-synthetic derivative of artemisinin and is rapidly converted in vivo to its active metabolite dihydroartemisinin (DHA). LFT is a long acting antimalarial drug, which is absorbed very slowly and has a longer active terminal half-life. The objective of the present work is to develop a selective and sensitive LC-MS/MS method to extract and quantify ARM, DHA and LFT simultaneously in plasma. Methods: Analysis was performed on an Agilent 1100 series HPLC system (Agilent Technologies, Germany) coupled to an AB Sciex API-4000 QqQ mass spectrometer (Sciex, Canada). All analytes were separated using an XBridge C18 (2.1 × 50 mm, 3.5 μm) column at 45 °C. The mass spectrometer operated in positive ion multiple reaction monitoring mode with transitions of m/z 316.2 → 267.2, m/z 302.3 → 267.2 and m/z 530.2 → 348.1 was used for detection of ARM, DHA and LFT, respectively. All the plasma samples were processed under ice cold conditions. After oral administration of single dose of artemether (4 mg/Kg) and lumefantrine (25 mg/Kg) to New Zealand White rabbits, the plasma samples were processed to quantitate ARM, DHA and LFT concentrations. Results: The most intense base peaks for ARM and DHA were produced by ammonium adduct [M+NH4]+, whereas, for LFT the base peak was produced by the protonated molecular ion [M+H]+ in ESI mode. The absence of interfering endogenous matrix components at the retention times of analytes demonstrated the specificity of the method. The mean extraction recoveries ranged from 94% to 98% for all analytes. Matrix factor as well as IS normalized matrix factor were within the accepted range of 0.9 – 1.1. Calibration curves were found to be linear across the ranges measured with regression coefficient greater than 0.998. All the analytes were found to be stable in plasma at room temperature (18 hrs), at -70 °C (38 days) and after 3 freeze-thaw cycles. In rabbits, ARM, DHA and LFT reached Cmax values of 2.7 ng/mL, 6.7 ng/mL and 427 ng/mL at Tmax of 15 min and 8 h, respectively. Conclusion: This is the first LC-MS/MS method reported for the simultaneous quantification of ARM, its active metabolite DHA and LFT in plasma. The PK results demonstrated that ARM and DHA exhibited rapid absorption and elimination, whereas, LFT has a long elimination half life. A rapid sample preparation procedure under controlled conditions of temperature prevented the degradation of artemisinin derivatives in plasma samples. The higher extraction recovery obtained with protein precipitation by organic solvent is advantageous when metabolites are quantified. This method is highly suitable for pediatric clinical trials where only small sample volumes are available.