Three-way partial last squares (3-PLS2), as a multi-way calibration method was applied for determining the 235U/238U isotope ratio to overcome problems with spectral interferences in liquid scintillation spectra of these radioisotopes. The alpha energy spectra of samples in different energy channels and different cocktail to sample ratios were used as input data for 3-PLS2. The model was applied to a prediction set and satisfactory results were obtained. The 3-way PLS2 prediction results were compared with 2-way PLS2 and it was shown that 3-way PLS2 results are more accurate than the results of PLS2. Thermal ionization-mass spectrometry was used as a reference method for calculating the accuracy of our method.