Summary The β‐Carotene (BC), an important precursor of vitamin A (VA), possesses antioxidant activity but is fat‐soluble and has low bioavailability. In previous in‐vitro assays evaluating antioxidant and 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) free radical scavenging, both BC and VA showed a strong ability to scavenge radicals and protected cells from oxidative stress. Here, we used artificially simulated gastrointestinal digestion and Caco‐2 cell absorption models to evaluate the bioavailability of the BC during gastrointestinal digestion and absorption using high‐performance liquid chromatography (HPLC) analysis. We observed high absorptive and transfer rates of BC and detected retinol metabolites (Vitamin A). Therefore, BC can be detected in the acidic gastrointestinal environment using HPLC. Optimised method provided better separation of BC and VA in the column, improving the accuracy of the test results.