Glycosaminoglycans (GAGs) play a crucial role in tissue homeostasis by regulating the activity and diffusion of bioactive molecules. Incorporating GAGs into biomaterials has emerged as a widely adopted strategy in medical applications, owing to their biocompatibility and ability to control the release of bioactive molecules. Nevertheless, immobilized GAGs on biomaterials can elicit distinct cellular responses compared to their soluble forms, underscoring the need to understand the This article is protected by copyright. All rights reserved. 2 interactions between GAG and bioactive molecules within engineered functional biomaterials. By controlling critical parameters such as GAG type, density, and sulfation, it becomes possible to precisely delineate GAG functions within a biomaterial context and to better mimic specific tissue properties, enabling tailored design of GAG-based biomaterials for specific medical applications. However, this requires access to pure and well-characterized GAG compounds, which remains challenging. This review focuses on different strategies for producing well-defined GAGs and explores high-throughput approaches employed to investigate GAG-growth factor interactions and to quantify cellular responses on GAG-based biomaterials. These automated methods hold considerable promise for improving our understanding of the diverse functions of GAGs. In perspectives, we encourage the scientific community to adopt a rational approach in designing GAG-based biomaterials, taking into account the in vivo properties of the targeted tissue for medical applications.