Abstract
Hyaluronic acid (HA) is a key extracellular biopolymer whose molecular weight (MW) critically determines its physicochemical behaviour and biological functionality. Accurate MW characterization is therefore essential for applications ranging from tissue engineering to medical and cosmetic formulations. Although previous reviews have addressed general aspects of HA, they have only briefly summarized analytical approaches for MW determination. In contrast, the present review provides the first dedicated and comparative analysis of the three most widely used and practically relevant techniques-agarose gel electrophoresis, size-exclusion chromatography coupled with HPLC (SEC-HPLC) and intrinsic viscosity measurements. We examine recent methodological advances, including improved calibration strategies, optimized gel matrices, multi-detector SEC configurations, and updated empirical models for viscometric analysis. Particular emphasis is placed on how these innovations enhance accuracy, reproducibility and applicability across different HA size ranges and sample purities. By critically evaluating the strengths, limitations and sources of analytical bias of each technique, this review offers a consolidated framework to guide researchers and industry professionals in selecting the most appropriate method for reliable MW assessment. Overall, we highlight how methodological refinement is enabling more robust characterization of HA, even in highly polydisperse or complex samples.