Proteins are molecules essential for the structure, function and regulation of almost all living organisms. These macromolecules are composed of amino acids connected by peptide bonds to form polypeptide chains that bend into a specific three-dimensional structure to create functional proteins. Determination of protein concentration in biological samples is crucial for understanding biological processes, for diagnostic purposes, and for the therapy of various diseases. The concentration of certain proteins outside the reference values may be associated with certain diseases or conditions. Quantification of total proteins in a sample is often the first step toward more complex analyses that then serve to better understand the biological functions of proteins and thus enable better treatment. The aim of this study was to determine whether the useof different analytical methods will affect the obtained value of protein concentration in biological tissue and serum samples. The concentration of total proteins was determined in brain, colon, and serum samples of C57BL/6 mice using four different approaches: bicinchoninic acid assay (BCA), bromphenol blue, Bradford and biuret methods. All of the aforementioned methods rely on the change in light absorption in the samples due to the interaction between the components of the reagent used and the protein of the sample. The light apsorption change is then measured spectrophotometrically. Statistical analysis showed that the results obtained by the Bradford method differed from those obtained by the other three methods for all three sample types, while there were no statistically significant differences between the BCA, the bromophenol blue, and the buiret method for any of the analyzed samples. According to our results, it can be concluded that the use of the BCA, bromophenol blue or buiret method for the determination of protein concentration in mouse serum, brain and intestine is an acceptable approach. The choice of the method used depends on the research objective, sensitivity, type and amount of the sample, as well as available resources. In evaluating the results of this study, it can be deduced that the combination of multiple methods provides more reliable characterization of proteins in biological samples. It is also important to pay attention to the freshness and maintenance of the prepared chemicals, as well as storage conditions of the biological samples to avoid false results.