The aim of the study was to determine serum free amino acid concentration in male dogs suffering from benign and malignant perianal tumours. Serum concentrations of cysteic acid, glutamic acid, glutamine, aspartic acid, glycine, γ-aminobutyric acid, tryptophan, methionine, arginine, taurine, threonine, tyrosine, alanine, valine, isoleucine, and leucine were significantly changed in dogs suffering from benign and/or malignant tumours when compared to the control group (P<0.05). Serum concentration of serine, phenylalanine, lysine and histidine was not influenced in dogs with neoplastic disease (P>0.05). The evaluation of serum free amino acid concentration, has shown that threonine, glutamic acid, alanine, valine, isoleucine, leucine, γ-aminobutyric acid, and glycine have the highest diagnostic and prognostic value in dogs suffering from benign and malignant perianal tumours. Highly diagnostic and prognostic value in relation to benign tumour growth was also found while evaluating branched chain and acidic groups of amino acids. Serine, phenylalanine, lysine, and histidine, as well as alkaline amino acids were proved not to have diagnostic and prognostic value in dogs with the tumours. Thus, the evaluation of free amino acid concentration may serve diagnostic purposes and help in tumour malignancy differentiation. The elaborated experimental model may be used in further studies on neoplastic disease development and monitoring of applied treatment efficiency.