Valproic acid (2-propylpentanoic acid) is an effective anticonvulsant widely used alone or in combination with other drugs (e.g., with carbamazepine, phenitoin, and phenobarbital) [1]. In the latter case, valproic acid (VA) decreases binding of other drugs (e.g., phenitoin and phenobarbital) to blood plasma proteins. On the other hand, phenitoin and phenobarbital also reduce the VA concentration in the blood plasma. VA is characterized by a high rate of absorption and elimination; for this reason the drug level in the plasma exhibits significant variations, especially if the interval between doses exceeds 8 h [2]. Therefore, monitoring of the VA level in the blood is very important both for successful therapy and for studying the VA pharmacokinetics and interactions with other drugs. Although numerous data have been reported on the determination of VA by various conventional methods [3 – 9], development of the new analytical procedures is still important for several reasons. First, VA, being a volatile compound, introduces large uncertainties as a result of losses in the sample concentration stage upon liquid extraction. Second, VA determination by HPLC with conventional UV detectors encounters problems related to weak optical absorption in this spectral range. For this reason, gas chromatography with a plasma-ionization detector was frequently preferred. However, being an acid, VA yields peaks with considerable tails on the GC chromatograms. This is usually eliminated by converting VA into derivatives (mostly by methylation) [3]. It should be noted that HPLC is still the most natural method for the analysis of biological samples, but VA has to be converted into derivatives exhibiting stronger absorption in the UV range, such as bromophenacyl, pentafluorobenzyl, or coumarin esters [4 – 9]. However, the release of VA in the course of derivatization (typically conducted in a micellar medium or upon precipitating proteins with acetonitrile [6, 9]) is incomplete because VA is strongly bound to proteins (80 – 90%) and is partly lost as a result of coprecipitation [6, 9]. Therefore, the total (bound plus free) VA concentration in the plasma is more reliably determined by methods involving the primary denaturation of proteins, followed by VA extraction with an appropriate organic solvent and, finally, derivatization [8]. The purpose of this study was to establish optimum conditions for VA extraction from blood plasma and conversion into bromophenacyl ester. We have also selected the optimum mobile and immobile phases for the HPLC analysis of bromophenacyl esters of VA (VABE) and nonanoic acid (NABE) and verified the method by determining VA in the blood plasma of experimental animals.