Slovak bryndza" is a traditional Slovak cheese manufactured by milling a lump of matured ewe's cheese or by milling a mixture of lump ewe's cheese and lump cow's cheese. The percentage of lump ewe's cheese is greater than 50% w/w dry matter. Differences in the purchase prices of ewe's and cow's milk create the potential for adulteration. The detection of cow's milk compounds by isoelectric focusing of γ-caseins after plasmolysis is a suitable method for the determination of adulteration of this traditional cheese. The analysis and final results may be affected by factors affecting laboratory activity and biological factors. Biological factors are manifested primarily as variations in the casein content of ewe's and cow's milk. Despite this variation, this method may also be used for quantification purposes. We were able to establish a reliable calibration line from the internal reference materials prepared in our laboratory. The mean composition of Slovak bryndza cheese determined in our research was protein 18.7 ± 1.9 g 100 g −1 , fat 24.0 ± 1.8 g 100 g −1 , lactose 1.5 ± 1.9 g 100 g −1 , ash 4.5 ± 0.3 g 100 g −1 , fat in dry matter 49.3 ± 2.6 g 100 g −1 , casein 18.4 ± 1.9 g 100 g −1 , gamma casein 0.60 ± 0.19 g 100 g −1, and bovine-to-ovine gamma casein ratio 0.65 ± 0.12 g 100 g −1 .