In general, antibiotics are used to treat infections. In some cases, antibiotic drugs are simultaneously in a sample and are used or injected simultaneously, thus controlling the amount of drugs in pharmaceutical products or their amount in biological samples is important. In this investigation, a novel net analyte signal standard addition method (NASSAM) was used for simultaneous determination of the drugs cefazoline and cefotaxim. The NASSAM can be applied for determination of analytes in the presence of known interferents. The proposed method is used to eliminate the calibration and prediction steps of multivariate calibration methods; the determination is carried out in a single step for each analyte. The accuracy of the predictions against the H-point standard addition method is independent of the shape of the analyte and interferent spectra. The net analyte signal concept was also used to calculate multivariate analytical figures of merit, such as LOD, selectivity, and sensitivity. The method was successfully applied to the simultaneous determination of cefazoline and cefotaxim in pharmaceutical formulations and spiked human blood serum samples.