A specific and robust method is presented for the determination of atovaquone in plasma. Atovaquone is a potent antiprotozoal compound for use in immunocompromised patients who are intolerant of conventional therapies. The method involves a liquid-liquid extraction of the compound into hexane modified with 2% (v/v) isoamyl alcohol. The processed extracts are analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 254 nm. The assay has a limit of quantification of 0.1 microgram/ml and is linear between 0.1 and 50 micrograms/ml. The method has been applied to many clinical studies and has been demonstrated to be precise and accurate with high sample throughput. Atovaquone is not significantly metabolised in humans.