A novel net analyte signal standard addition method (NASSAM) was used for simultaneous determination of the drugs anthazoline and naphazoline. 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 anthazoline and naphazoline in a commercial eye drop sample.