Black cumin (Nigella sativa) seeds are a rich source of thymoquinone, a bioactive compound with numerous therapeutic properties. Accurate quantification of thymoquinone is crucial for quality control and standardization of black cumin seed-based products. This study presents a rapid and reliable high-performance thin-layer chromatography method for the quantitative analysis of thymoquinone in black cumin seeds. The method involves a simple extraction procedure followed by HPTLC separation and densitometric detection. Key parameters such as mobile phase composition, developing distance, and detection wavelength were optimized to achieve optimal separation and sensitivity. The developed HPTLC method demonstrated good linearity, precision, accuracy, and sensitivity, making it suitable for routine analysis of thymoquinone in black cumin seeds and related products.