This article analyzes the sources, production methods, and biomedical potential of chondroitin sulfate (CS), with a particular emphasis on its extraction from byproducts of freshwater fish processing. CS, as a sulfated glycosaminoglycan, is a key structural and functional component of the extracellular matrix, playing a critical role in maintaining joint health, neuroplasticity, tissue regeneration, and modulating inflammatory processes. Its biological activity directly depends on structural features such as the degree of sulfation ("sulfate code"), molecular weight, and spatial organization, which, in turn, are determined by the source of the raw material and the extraction technology. The paper emphasizes that traditional industrial production of CS relies on animal-based raw materials (cartilage from cattle, pigs, and chickens), which is associated with a number of limitations, including religious considerations, the risk of zoonotic diseases, and the instability of the raw material base. In this context, aquatic organisms, and particularly by-products of the fish processing industry (skeletons, cartilage, heads), are considered a promising and sustainable alternative. The use of such raw materials solves the problem of waste disposal and is consistent with the principles of a circular economy. This article provides a detailed review of traditional (alkaline and acid hydrolysis) and modern (enzymatic, ultrasonic, microwave, supercritical fluid extraction) methods for extracting cholesterol. It is concluded that the development of effective and cost-effective technologies for obtaining cholesterol from fish waste is a pressing scientific and practical challenge. Such technologies must ensure not only high yield and purity but also preserve the specific structure of sulfated isoforms, which determines their therapeutic efficacy. The implementation of such solutions will expand the raw material base for cholesterol production, create new value chains in the fishing industry, and meet the growing market demand for functional nutraceuticals and biomedical materials with predictable properties.

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