Quality control of herbal medicines is an important stage in generating effective and safe herbal remedies. A unique high-throughput approach for assessing anti-diabetic phytoconstituents in Syzygium cumini seed extract and marketed formulation was examined using high-performance thin-layer chromatography (HPTLC)–bioautography technology. Using β-sitosterol as reference, this agar-free bioautographic technique was devised and validated for the assessment of α-amylase inhibitors on TLC plates. The presence of some anti-diabetic compounds in plant samples was verified using a combination of biological detection and mass spectrometry (HPTLC–MS). The newly proposed HPTLC–bioautography screening method may be effective for evaluating the anti-diabetic activity of plant extracts and formulations. The proposed technique is quick, easy, precise, and dependable.