eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2026-03-30 23 1 221 232 10.13005/bbra/3492 58379 article Pravin Rangnath Dighe 1 Manoj Ramesh Kumbhare 1 Department of Pharmaceutical Chemistry, S.M.B.T. College of Pharmacy, Affiliated to Savitribai Phule Pune University, Maharashtra, India. The present work aimed to develop a bioanalytical RP-HPLC technique for the simultaneous quantification of brexpiprazole (BREX) and fluoxetine hydrochloride (FLX) in human plasma. The chromatographic separation was performed on a Phenomenex C18 column (250 mm × 4.6 mm, 5 µm). The chromatogram was recorded at 224 nm. As an internal standard (IS), fimasartan was used. The mobile phase was composed of acetonitrile and 0.05% orthophosphoric acid in water (35:65), and with a flow rate of 1.0 mL/min. Separation was carried out in an isocratic mode. The protein precipitation method was used to isolate the analytes from the spiked plasma matrix. The retention times (RT) of BREX, IS, and FLX were 3.69, 4.91, and 6.82 min, respectively. With regression coefficients r² = 0.9996 and 0.9993 for BREX and FLX, respectively. The developed method demonstrated an acceptable linearity in the concentration range of 1.40–56 µg/mL. The extraction recovery of BREX was in the range of 82.57% to 85.26%, and for FLX, 91.74% to 94.05%. The stability studies showed no evidence of analyte degradation. Hence, the developed method can be used for the simultaneous quantification of BREX and FLX in biological samples. https://www.biotech-asia.org/vol23no1/bioanalytical-method-development-and-validation-for-simultaneous-estimation-of-brexpiprazole-and-fluoxetine-hydrochloride-in-human-plasma-by-rp-hplc/ Brexpiprazole; Bioanalytical method validation; Fluoxetine hydrochloride; Mood disorders; Protein precipitation; Serotonin transporter