Type 2 diabetes mellitus is a chronic metabolic disorder which is mainly correlated to hyperglycemia, hyperlipidemia, oxidative stress, and protein glycation. Bioactive compounds from herbal resources suggest a dietary approach for diabetes management by inhibiting carbohydrate-degrading enzymes and biomolecules oxidation. The anti-diabetic activities and amino acids composition of four medicinal plants, including Oliveria decumbens, Thymus kotschyanus, Trachyspermum ammi, and Zataria multiflora, were investigated. Free and protein amino acid extracts are mainly composed of amino acid and lower levels of monosaccharide, fatty acid, phenol compound, flavonoid, and tannins. The ratio of phenol/amino acid, flavonoid/amino acid, and tannins/amino acid in free amino acid extract was higher than protein amino acid extract. Liquid chromatography–mass spectrometry characterization showed that the main free amino acids among the plants analyzed in this study were asparagine (0.12–3.67 g/100 g), arginine (0.42–1.31 g/100 g), beta-aminoisobutyric acid (0.33–0.94 g/100 g), glutamine (0.20–0.54 g/100 g), aspartic acid (0.05–0.91 g/100 g), glutamic acid (0.07–0.49 g/100 g), proline (0.05–0.49 g/100 g), alanine (0.20–0.33 g/100 g), phenylalanine (0.04–0.32 g/100 g), valine (0.05–0.24 g/100 g), leucine (0.05–0.23 g/100 g), and serine (0.03–0.24 g/100 g). The main amino acids from plant-derived protein hydrolysate were aspartic acid (1.4–5.39 g/100 g), glycine (1.64–4.25 g/100 g), glutamic acid (1.37–3.93 g/100 g), leucine (1.35–2.92 g/100 g), alanine (1.45–2.79 g/100 g), lysine (1.38–2.67 g/100 g), arginine (1.33–2.97 g/100 g), serine (1.47–1.90 g/100 g), phenylalanine (0.79–1.96 g/100 g), valine (0.69–1.47 g/100 g), proline (0.84–1.25 g/100 g), and histidine (0.68–1.46 g/100 g). Free amino acid extract and protein amino acid extract exhibited moderate anti-glucose oxidation, anti-lipid oxidation, anti-protein oxidation, and anti-protein glycation, and anti-amylase, and anti-glucosidase activities. Antioxidant and anti-diabetic properties of free amino acids were considerably higher than the protein-hydrolyzed amino acids that may be attributed to higher phenol/amino acid, flavonoid/amino acid, and tannins/amino acid ratios.