In this paper, some up-to-date methods of protein separation and purification appearing useful in research and food analysis were reviewed. The protein purification process includes four major phases: selecting a source of protein, isolating and extracting protein from the biological material, purifying the extracted protein using a column chromatography technique, and, finally, storing the protein obtained until it is taken for further analysis. The content of protein in a biological material and its distribution is one of the factors determining the protein source to be selected. It is possible to win considerable amounts of proteins by employing a DNA recombination technique leading to their increased expression in bacterial cells. When isolating intracellular proteins, it is necessary to utilize additional measures in order to destroy both the tissues and the cells. The cells present in a suspension are destroyed using several methods. With regard to integral protein bound to a biological membrane, an anionic detergent, for example Tryton X-100, is applied to such a biological membrane since this anionic detergent does not cause the denaturation of protein, and it prevents inactivation of the proteins. Prior to starting the purification process of proteins, it is necessary to determine the application of the final proteins to be obtained, the required degree of their purity, and their activity. Depending on its applications, the final protein product can have three degrees of purity: very high > 99%, high, ranging between 95 and 99%, and moderate < 95%. Chromatographic techniques are employed to separate macromolecules on the basis of such parameters like: size and shape, hydrophobicity, surface net charge, or affinity. The first of those techniques is molecular sieving chromatography, also known as a gel filtration. If this technique is applied, macromolecules are separated according to their sizes and shapes. The other technique is ion exchange chromatography and with it, proteins are separated according to differences in the surface net charge of protein macromolecules. While applying the hydrophobic interaction chromatography, the separation of proteins is based on differences in the protein hydrophobicity, whereas the affinity chromatography utilizes the affinity of two substances, for example an enzyme and a substrate, and one of these two substances is carrier-bound, thus, immobilized. In the paper, the purity degree of protein as a final product was discussed with regard to the potential application of such a protein product.