<p>Cellulase is an essential microbial enzyme involved in the degradation of cellulose; a common polysaccharide present in many animal feedstocks. Currently, known cellulase activity measurement methods have drawbacks, including the use of toxic chemicals (3,5-dinitrosalicylic acid, DNS) and/or complex reagent compositions. This study reports an environmentally friendly spectrophotometric method for rapidly determining cellulase activity using the CUPRAC reagent (Cu(Nc)₂²⁺). Additionally, more ecologically friendly and simplified alternatives have been developed, such as the CUPRAC-Cellulase protocol, which evaluates the formation of a colored Cu(I)-neocuproine complex at 450 nm, directly related to glucose formation as a product of cellulose hydrolysis. The CUPRAC-Cellulase assay demonstrated good sensitivity (r = 0.99) compared to reference methods. The method exhibited a linear range between 0.05 and 5.0 FPU/mL, and the determination varied little with several biological samples tested. The intra-day and inter-day coefficients of variation (CVs) were 2.2 and 4.5%, respectively, indicating excellent precision of the method. Moreover, the matrix effect was significantly reduced, resulting in improved glucose specificity and reduced interference from other biomolecules. The CUPRAC-based assay is a rapid and environmentally safe alternative for determining cellulase activity. Its capability of removing toxic reagents and simplifying the assay procedure makes it an excellent choice for measuring cellulase, both in research and industry. This novel method enables accurate comparisons to be made on cellulase efficacy in different biological scenarios.</p> Graphical abstract <p></p>