β-carotene as a dietary supplement is a matter of interest to anyone who seeks to improve their skin condition or sight. Due to its antioxidative properties, carotenoids are involved in the prevention of severe conditions, such as cancer, heart disease, macular degeneration, or cataract, which makes them an object of scientific study. Thanks to its color, ß-carotene is used as a food colorant, improving the appearance of foodstuff. A presented method was established for the identification and quantification of β-carotene by thin-layer chromatography with densitometric detection. As a stationary phase, TLC Aluminiumoxid 60 F 254 neutral was utilized. It were activated with methanol and dried at the temperature of 60°C for 1 h. The development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of chloroform/methanol/acetone/ammonium hydroxide (10:22:53:0.2, v/v/v/v). TLC scanner was used for densitometric scanning and analysis in the absorbance mode at 450 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). Presented method illustrates simple, economical, and suitable manner for routine quantitative and qualitative determination of β-carotene in dietary supplements and carrot juice.