Concentrations of the anthelmintic agent levamisole were measured in a total of 85 raw cow milk samples collected during 2014 from dairy farms across Croatia. The liquid chromatographytandem mass spectrometry (LC-MS/MS) method used for levamisole quantification was validated according to the criteria set by Commission Decision 2002/657/EC. The following validation parameters were determined: limit of decision (CCα) 0.55 μg kg-1, detection of capability (CCβ) 0.59 μg kg-1, limit of detection (LOD) 0.06 μg kg-1, limit of quantification (LOQ) 0.22 μg kg-1, precision and accuracy (expressed as recovery) 97.3-100 %, intra-laboratory reproducibility (RSD) 4.2-5.6 %. Levamisole levels for 45 of the total of 85 samples were below the LOD value (0.06 µg kg-1). In the remaining 40 milk samples, levamisole was measured in the range from 0.061 to 0.142 µg kg-1 and the mean value was 0.092 µg kg-1. Accordingly, all concentrations analysed were below the LOQ value (0.22 µg kg-1) and limit of decision (CCα) of the method used.