Picroliv is a new hepatoprotective agent, being developed at the Central Drug Research Institute (CDRI, Lucknow, India). Picroside I is a major active constituent of picroliv. A sensitive high-performance liquid chromatographic assay method in plasma has been developed and validated for the determination of picroside I in plasma. The method consists of extraction of the drug, after protein precipitation with methanol, from rabbit plasma samples. Separation was achieved using a C18 endcapped reversed-phase column coupled with a photodiode array detector and acetonitrile-0.1 M acetic acid (25:75) as mobile phase. The lower limit of quantification in plasma is 50 ng/ml. The standard curve was linear over the range of 50-500 ng/ml in rabbit plasma. The analytical recovery of picroside I added to plasma was >80%. The reproducibility was determined by the inter- and intra-assay precision which were <15%.