Medicinal plants and their combinations due to the wide range of biologically active substances can influence on various links of the pathogenetic mechanism of development of DM type 2 and its complications. One of such combinations is an antidiabetic herbal mixture (Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices) with established hypoglycaemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective activity in previous pharmacological studies in vivo and in vitro and defined phytochemical composition. Thus, the aim of this study was to identify and establish the content of amino acids in the plant components of antidiabetic herbal mixture. The amino acids were separated by GC-MS method with pre-column derivatization. The calibration curves of twenty CRS of amino acids were linear (R2 > 0.98) over the range of 1–100 µg/mL, the LODs and the LOQs were in the range of 0.01–0.07 µg/mL and 0.02–0.20 µg/mL, respectively. The results of analysis showed that the predominant essential amino acid was L-proline in Taraxaci radices, Urticae folia, Rosae fructus and Menthae folia, its total content was 101.46 mg/g, 25.31 mg/g, 23.04 mg/g and 19.30 mg/g, respectively. In addition, it was established total content of essential amino acid – L-leucine that can stimulate insulin secretion in β-cells of the pancreas. Its total content was 58.51 mg/g in Taraxaci radices, 9.58 mg/g in Myrtilli folia, 4.68 mg/g in Rosae fructus, 2.99 mg/g in Urticae folia and 0.79 mg/g in Menthae folia. Chromatographic examination also revealed L-phenylalanine, an essential amino acid important for antidiabetic therapy that can increase insulin secretion, stimulate proliferation and neogenesis of β-cells of the pancreas and reduce insulin resistance. Its total content was 13.42 mg/g in Myrtilli folia, 2.23 mg/g in Rosae fructus, 1.478 mg/g in Urticae folia, 1.46 mg/g in Taraxaci radices and 0.52 mg/g in Menthae folia. This phytochemical study shows, which plant material forms the amino acid composition and content in the finished herbal mixture and due to which biologically active substances the antidiabetic activity of this phytocomposition is manifested.