An analytical methodology appropriate for the determination of the novel drug candidate salicylaldehyde isonicotinoyl hydrazone (SIH) in rabbit plasma has been developed and validated. Desirable chromatographic separation was achieved on a C18 column employing a mixture of phosphate buffer (0.01 M NaH2PO4 x 2 H2O with 2 mM EDTA, pH 6.0) and methanol (53:47; v/v) as the mobile phase. In order to develop a suitable sample preparation procedure, different methods have been tested (solid-phase extraction, liquid-liquid extraction, and protein precipitation). Protein precipitation using 0.1 M HClO4 and acetonitrile allowed the highest recoveries of the analyte to be reproducibly attained. The analytical methodology developed in this study was validated with respect to linearity (0.26-30.0 microg/mL), accuracy, precision, selectivity, recovery, and stability. A concentration of 0.26 microg/mL was determined as the LLOQ. The chromatographic method was applied to a preliminary plasma pharmacokinetic study. This study has provided the first information about the concentrations of SIH in plasma of a living subject. These results could have a significant impact on further progress in the development of this promising compound.