The UV spectrophotometric analysis of a binary mixture containing bisoprolol fumarate (BS) and hydrochlorothiazide (HZ) by using multivariate calibration methods, such as principal component regression (PCR) and partial least-squares regression (PLS-1); and a graphical spectrophotometric method such as second derivative of the ratio spectra 2 DD was described. A calibration set of 22 reference samples was used to develop the models for PCR and PLS-1. The calibration models optimization was achieved through the proper wavelength ranges selection, and excluding any information about the interfering excipients. In addition, a High Performance Liquid Chromatography (HPLC) was developed to analyze the same mixture. The chromatographic separation was achieved on a reversed phase, RP 18 column with mobile phase consisting of acetonitrile-0.01 M KH2PO4 (40:60, v/v, and pH 3.5). Quantitation was achieved with UV detection at 232 nm based on peak area. The proposed methods were validated and successfully used for the analysis of laboratory-prepared mixtures and pharmaceutical formulations.