High performance liquid chromatography with UV/vis detection was optimized and validated for simultaneous quantification of alprazolam with celecoxib and diclofenac sodium in pharmaceutical formulation and human serum. Chromatographic separation was achieved at detection wavelength of 230 nm on Shimadzu Shim-pack CLC-ODS (M) 25M column employing 80:20 (v/v) methanol: water (pH 3.5) as mobile phase with elution rate 1.0mL min-1. Analytes were quantified in the ranges 0.2-15, 0.3-20 and 0.6-40 µg mL-1 with detection limits 19.76, 17.29 and 11.83ng mL-1 respectively. Recoveries were in the range 98.15-101.15, 99.24-99.90 and 98.87-101.19% in pharmaceutical formulation and 98.05-101.01, 98.72-99.49 and 98.25-99.47% in human serum respectively and precision ranged from 0.19-1.84%. The analytes were successfully detected without any observable interference commonly present in pharmaceutical formulation and human serum demonstrating applicability of method.