Supercritical fluid extraction (SFE) has been widely used for extracting several valuable phytochemicals, including carotenoids. However, there is a scarcity of works dealing with the purification of SFE extracts. The aim of this work was to assess the feasibility and efficiency of a hydrophobic interaction chromatography (HIC) protocol for purifying carotenoid-rich extracts obtained by SFE. Initial batch experiments were carried out to calculate the resin adsorption capacity and adsorption kinetics. Subsequent runs were performed in a manually packed chromatographic column, using the Amberlite XAD-1180N resin, where breakthrough curves and adsorption isotherms were obtained and fitted to the Langmuir model. The antioxidant activity and carotenoid degradation rates were monitored throughout the processes. In batch, the resin presented a maximum carotenoid adsorption capacity of 1.89 g/mg, while in column, this value increased to 10.4 g/mg. The global carotenoid adsorption rate was 93.3% and the elution rate, 94.7%, resulting in a global recovery of 88.4% for total carotenoids and 92.1% for carotenes. The Langmuir model fitted well the experimental data. Analysis of the extracts demonstrated that a 5.5-fold reduction in extract mass was achieved, accompanied by a 4.7-fold and 2.1-fold increase in carotenoid concentration and antioxidant activity, respectively. This work presents a novel process based on preparative HIC for the purification of carotenoid extracts and provides a fundamental understanding on process performance. It is potentially scalable and can be implemented in extraction and purification of carotenoids from natural sources, as an alternative to their production through chemical synthesis.