An accurate, reliable, specific and sensitive spectrofluorimetric method was developed for the determination of ampicillin in the presence of alizarin. The method is based on the reaction between ampicillin and alizarin in aqueous solution to form a complex with violet color. UV-vis and spectroflourimetry techniques were applied in this research. The affecting factors such as reaction time, pH of the solution and the mole ratio of ampicillin to alizarin were examined. The UV-vis and fluorescence spectra of the ampicillin in the absence and presence of alizarin were recorded. The fluorescence spectra of ampicillin were recorded with the excitation wavelength (ex) of 334 nm. Results indicated that band intensity of the fluorescence spectrum for ampicillin is considerably enhanced in the presence of alizarin, this phenomenon leads to improve detection limit as well dynamic linear range. The calibration curves were constructed using solutions with different concentrations of ampicillin in the presence and absence of alizarin. Detection limit were obtained 024 to 0.11 ppm using UV-vis spectroscopy and 0.05 to 0.003 ppm using spectrofluorimetry. The concentration of ampicillin was determined in real sample (ampoule) using standard addition method. The interferences were also studied to find selectivity method. In the second part of this work, the multivariate calibration, together with partial least-squares (PLS-1), principal component regression (PCR) and hybrid linear analysis (HLA) models were applied for simultaneous measurement of in solution containing three drugs of amoxicillin, metronidazole and omeprazole. Data of analysis were obtained from recording of UV–vis spectra of the solutions. The experimental and theoretical data were compered and showed that the theoretical data are good agreement with the experimental values with low relative error percentage. Results indicated that the HLA model is more suitable than the other models. In this respect, the relative error was obtained for amoxicillin, metronidazole and omeprazole 0.09 %, 0.7% and 0.8% respectively. The procedure was successfully applied for simultaneous determination of the above drugs in pharmaceutical tablets,
