A simple, sensitive, selective, rapid, rugged, reproducible and specific Ultra performance liquid chromatography (UPLC) method was used for a quantitative estimation of Atovaquone (AQ) in Rat plasma using 2,3-diphenyl-1- indenone (AQIS) as an internal standard (IS). Chromatographic separation was performed on Waters Acquity UPLC® BEH C18, 2.1 x 50 mm, 1.7 μm column with an isocratic mobile phase composed of 0.1% formic acid : methanol (20:80 v/v), at a flow-rate of 0.2 mL/min AQ and AQIS were detected with waters TUV detector at UV wave length 277 (nm). Liquid-liquid extraction method was used and validated over a linear concentration range of 50.0-10000.0 ng/ mL with a correlation coefficient of (r2) ≥ 0.9981. The lower limit of quantification (LOQ), Limit of detection (LOD) was found as 50.0 ng/ mL and 100 p.g respectively. Intra and inter-day precision within 1.0 to 8.5 and 1.9 to 4.3 % and accuracy within 101.0 to 107.5 and 103.9 to 104.7 % for AQ. Drug found to be stable throughout three freeze-thaw cycles. This method was successfully applied into a pharmacokinetic study of rats through i.v administration.
