Three independent reversed phase high-performance liquid chromatography (HPLC) procedures with diode array detection (DAD) for the analysis of carbamazepine (CBZ), topiramate (TPM), and valproic acid (VPA) have been developed in order to determine drug penetration of the blood—brain barrier. Determination of CBZ was performed on C18 column with mobile phases containing methanol (55%, v/v), acetate buffer at pH 3.5 (20%, v/v), double distilled water (25%, v/v), and 0.025 M L−1 diethylamine (DEA). The mobile phase containing acetonitrile and water (8:2, v/v) or acetonitrile and phosphate—citrate buffer at pH 2.6 (1:1), respectively, for analysis of VPA and TPM was applied. Quantification of carbamazepine was performed at 285 nm without extraction procedure before the analysis. Determination of topiramate and valproic acid was performed using precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). FMOC-Cl is a suitable agent, which reacts with both primary and secondary amines and also with acidic groups. Topiramate was determined at 263 nm and valproic acid at 300 nm. The proposed procedures are simple, not time-consuming, and suitable for the determination of investigated compounds in mouse brain homogenates.