HPLC separation of carotenoids eluted on C30 column as found in the following order; epoxy-carotenoids, hydroxy-carotenoids, and hydrocarbon carotenoids. Capsanthin and capsorubin were detected as characteristic pigments in the red pepper. The optimized HPLC condition separated and quantified carotenoids. The mass spectra of each carotenoid confirm by its characteristic ions. The mass spectra signals for epoxy-carotenoids are characterized by [M + H]+; m/z 601.3], [M + H]+; m/z 584.6] for capsanthin, loss of the water (lutein) or no loss of water (zeaxanthin) moiety from the respective quasimolecular ions representing the lutein backbone ([M + H - H2O]+; m/z 551) and β-carotene [M + H]+; m/z 536.8] The capsanthin rich fraction eluted on silica column yielded 92 ± 3% purity. The APCI+ve detector parameters adjusted suitably to give ion signal intensity for identification of peaks using respective standards. Red pepper digested sample fractioned larger particle-sized micelles than only carotenoids and carotenoids+capsaicin solubilized micelles. The PDI of carotenoids+capsaicin indicated the narrow distribution of particle size and stable zeta potential than other groups. The viscosity of carotenoids+capsaicin was lower than red pepper, and only carotenoids digested derived micelles. Smaller particle size, stable zeta potential, uniformly dispersed, and less viscous micelles found suitable for improving bioaccessibility/bioavailability of carotenoids at the gastrointestinal region. Therefore, increased bioaccessibility of carotenoids observed in capsaicin treatments than the sample without capsaicin. These results suggested that co-consumption of phytoconstituents with carotenoid-rich diet affected bioavailability of carotenoids. Also, understanding its regulatory mechanism at the intestinal level is important for the enteral nutrition of carotenoids.