This study reports the development and validation of a sensitive and selective assay method for the determination of alpha-,beta-arteether in rat serum by liquid chromatography-mass spectrometry. The mobile phase was composed of methanol-0.1 mM sodium acetate (pH 5) (80:20%) at a flow-rate of 1 ml min(-1) and chromatographic separations were achieved on a Ultracarb, 5 ODS 20, Phenomenex column (5 micrometer, 30 mmx4.6 mm I.D.). The total effluent from the column was split so that one-tenth was injected into the electrospray LC-MS interface. ESI-MS analysis was carried out using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at a cone voltage of 52 V with a scan range of 100-400 Da. The analytes were quantified from the [M+Na](+) ion chromatograms of alpha-,beta-arteether at m/z 335 and artemisinin at m/z 305. A simple liquid-liquid extraction with 2x2 ml n-hexane was used to isolate alpha-,beta-arteether from rat serum. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recovery from spiked control samples ranged from 88.41 to 96.17% with a maximum CV of 10.8% for alpha-arteether and 69.83-79.69% with a maximum CV of 17.06% for beta-arteether. Linearity in serum was observed over the range 20-320 ng ml(-1). Percent bias (accuracy) was well within the acceptable range. Within- and between-assay precision were less than 15%. The assay method described here is being applied to study the pharmacokinetics of CDRI developed intramuscular formulation Emal (alpha-/beta-arteether in the ratio of 30:70) in rats. The method is sensitive enough to monitor alpha-,beta-arteether up to 24 h after a single 30 mg kg(-1) i.m. dose.