Industrial scale manufacture of pharmaceutical grade hyaluronic acid increasingly uses a recombinant source of hyaluronidase to achieve size-specific polymers, necessary for both cosmetic and medical purposes. A turbidimetric assay is currently recommended by the United States Pharmacopeia (USP) to measure specific hyaluronidase enzyme activity. However, this monograph lacks critical detail of experimental methodology, uses difficult to source reagents and is technically challenging to perform. Herein, a simplified assay to measure the specific activity of hyaluronidase with improved reliability, consistency and flexibility is presented. Results demonstrated that the source and concentration of substrate significantly influenced turbidity formation, which occurred only under acidic conditions and when measured by absorbance at 400 nm rather than 640 nm. Replacement of horse serum as the binding protein agent with bovine serum albumin enhanced accuracy and precision, consistently meeting acceptance criteria. The improved protocol also introduced flexible timing, enabling reliable measurements within a 30 mins to 1 h window, compared to a fixed 30-minute time point. Inference by different compounds at concentrations normally used in standard buffers and fermentation media was not significant. However, Tris-HCl and Na2HPO4 significantly reduced the specific activity of hyaluronidase and are, therefore, not recommended in growth media for yeast cultivation, buffers used for enzyme purification, and in reagents necessary for monitoring production of size-specific hyaluronic acid polymers. A robust assay that can be validated is an important part of any enzyme dependent manufacturing process and based on our empirical experimentation, hereby propose revision of the existing USP monograph.