Liquid scintillation counting (LSC) is a commonly used technique for the determination of alpha and beta emitters. However, LSC has poor resolution and the continuous spectra for beta emitters hinder the simultaneous determination of several alpha and beta emitters from the same spectrum. In this paper, the feasibility of multivariate calibration by partial least squares (PLS) models for the determination of several alpha (natU, 241Am and 226Ra) and beta emitters (40K, 60Co, 90Sr/90Y, 134Cs and 137Cs) in water samples is reported. A set of alpha and beta spectra from radionuclide calibration standards were used to construct three PLS models. Experimentally mixed radionuclides and intercomparision materials were used to validate the models. The results had a maximum relative bias of 25% when all the radionuclides in the sample were included in the calibration set; otherwise the relative bias was over 100% for some radionuclides. The results obtained show that LSC-PLS is a useful approach for the simultaneous determination of alpha and beta emitters in multi-radionuclide samples. However, to obtain useful results, it is important to include all the radionuclides expected in the studied scenario in the calibration set.