Dataset containing analysis of free serum amino acid concentrations in patients with head and neck spinocellular tumors. The study was conducted in accord with the Helsinki Declaration of 1964 and all subsequent revisions thereof. It was approved by the ethical committee of St. Anne’s Faculty Hospital, Brno, and by ethical committee of University Hospital Motol, Prague, Czech Republic. All blood samples were obtained from HNSCC patients who developed histologically verified primary HNSCC after they signed the informed consent. Blood samples were obtained by venipuncture. The blood samples were centrifuged at 3000 rpm at 4°C for 10 min within 60 min after collection. Serum was aliquoted and stored at −80°C until analysis. The inclusion criteria were: age 40-95 years; no prior chemotherapy; no endocrinologic or metabolic disorders; no uncontrolled hypertension or infections; normal liver, heart and kidney function; and adequate bone marrow reserve. Amino acid profiles were examined using ion-exchange liquid chromatography (AAA-400, Ingos, Prague, Czech Republic) with post-column derivatization by ninhydrin and absorbance detector in visible light range (IEC-Vis).