A sensitive and specific liquid chromatographic method using extraction with zinc sulfate has been developed for the determination of Casiopeina IIIi and validated over the linear range 5-100 microg/ml in 1 ml of rat plasma. The analysis was performed on a Symmetry C(18) (5 microm) column. The mobile phase was methanol: 0.01 M phosphate buffer pH 6.5 (40:60, v/v). The column effluent was monitored at 262 nm. The results showed that the assay is sensitive at 5 microg/ml. Maximum intra-day coefficient of variation was 10.6%. The recovery obtained in plasma was 87.2%. The method was used to perform protein binding studies by equilibrium dialysis in rat plasma and was found to be satisfactory.