A rapid and simple high-performance liquid chromatography (HPLC) method using extraction with zinc sulfate has been developed for the determination of Casiopeina III-ia (Cas III-ia) and validated over the linear range 10-120 g/mL in 200 L of rabbit blood. Previously we have reported a HPLC method to quantify CasIII-ia in plasma, but in distribution studies in total blood [20], the Cas III-ia concentrations were higher in total blood that in plasma [19]. The analysis was performed on a Symmetry C (18) (5 microm) column. The mobile phase was Methanol-sodium phosphate buffer (pH 6.5; 0.01 M) (40:60 v/v) was kept at a flow-rate of 0.8 ml/min. The analyses were performed at room temperature. The column effluent was monitored at 262 nm. Acetaminophen was used as internal standard. The results showed that the assay is sensitive at 10 g/mL. Maximum intra-day coefficient of variation was 5.10%. The average recovery obtained in blood was 94.51%. The applicability of this method for distribution in vitro and pharmacokinetic studies in rabbits was demonstrated. Conclusions: the present assay is rapid, simple, precise, and accurate. The pharmacokinetic study was carried out in rabbits and the following pharmacokinetic parameters were obtained: (kel) = 0.0150 min-1, half-life time (T1/2) = 53.92 min, apparent volume of distribution (Vd) = 202.81 mL, clearance (Cl) = 2.08 mL /mi and area under the curve (AUC)=23163.85 µg/mL.min. Contributing to the preclinical characterization of the Cas III-ia.