The extraction of carotenoids from algae is currently the focus of extensive study, driven by the increasing demand for naturally occurring chemicals that are particularly abundant in physiologically bioactive compounds. Natural carotenoids exhibit superior stability compared to their synthetic counterparts, which have wide-ranging applications in the food, drug, and nutraceutical industries. Traditional liquid extraction is hindered by several disadvantages, including the requirement for substantial solvent quantities and multiple extraction stages. This protocol chapter delineates a comprehensive approach for the enzymatic hydrolysis of microalgal cell walls to improve carotenoid extraction, accompanied by systematic parameter adjustment to maximize yield and purity. The procedure delineates the selection and preparation of appropriate enzymes, the design of hydrolysis experiments employing statistical optimization methods like response surface methodology (RSM), and the extraction and measurement of carotenoids utilizing conventional analytical techniques. Essential procedures for enhancing the process are provided to promote repeatability and practical implementation. This chapter intends to function as an exhaustive resource for researchers and practitioners engaged in the sustainable and effective extraction of carotenoids from microalgal biomass.