Introduction: Health, religious, and commercial aspects justify the need for meat species identification. The lack of officially approved methods prompts the undertaking of research on validation of isoelectric focusing of proteins (IEF) for official purposes. Material and Methods: Samples were prepared from pigs (Sus scrofa ferus domestica), cattle (Bos taurus), and poultry (Gallus gallus domesticus). Meat mixtures were made by blending 50%, 25%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2% meat of other species. Samples were examined on ultrathin polyacrylamide gels with pH 3–9 gradient. Results: The results of the study confirmed the stable and reproducible pattern of meat protein bands. The detection limit of raw meat admixtures from pigs, cattle, and poultry mostly ranged from 2% down to 0.2% (0.2% for poultry). However, the IEF method can be used to detect the addition of pig meat to bovine meat in an amount higher than 3%. At the significant mixture level (i.e. at least 5% addition of meat of another species) IEF proves itself with 100% specificity, sensitivity, and accuracy. Conclusion: The achieved detection limits provide a basis for recommending the IEF method for routine tests in laboratories detecting the species origin of meat.