School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia E-mail : yusrida@usm.my Fax : 6 04 6570017 Manuscript received online 03 September 2014, revised 08 March 2015, accepted 13 March 2015 A new simple, sensitive and specific high performance liquid chromatography with UV detection (HPLC-UV) method was developed and validated for determination of Atovaquone in rabbit plasma. The method utilized 250 µL of plasma and Atovaquone was extracted from the plasma by using liquid-liquid extraction method. Buparvaquone was used as the internal standard and the method was validated on a C18 column using acetonitrile-ammonium acetate (pH 3; 0.02 M) (85 : 15, v/v) with UV detection at 254 nm. The assay was conducted at 45 ºC and a flow rate of 1 ml/min. The calibration curve was linear between 80–4000 ng/ml (r2 = 0.9993) with a limit of quantification (LOQ) of 80 ng/ ml. The extraction recovery ranged from 84.5 to 97.9% and plasma samples containing Atovaquone were found to be stable in –20 ºC for 4 weeks. Finally, the validated HPLC-UV method was successfully applied in an in vivo pharmacokinetic study involving New Zealand rabbits.