Keratin, in the form of coarse sheep wool, has been identified as an undervalued natural resource, which with the appropriate tools (e.g. a keratinase biocatalyst) can be repurposed for various textile and industrial biotechnology applications. For these purposes, we describe a novel method for identifying keratinase activity through the use of α-keratin azure (KA), an anthraquinone dyed substrate. A colourimetric method monitored the keratinase activity of Proteinase K (PK), which degrades the KA substrate and releases soluble products that are observed at 595 nm. Initially, the azure dye standard, Remazol Brilliant Blue R (RBBR), was used to calibrate the assay and allowed the kinetics of the keratinase-catalysed reaction to be determined. The assay was also used to investigate substrate pre-treatment, as well as different reaction quenching/work up conditions. Milling and washing of the KA substrate provided the best reproducibility and centrifugation was the most effective method for removing unreacted starting material. This assay was then applied to investigate the reduction of the keratin disulfide bond on keratinase-catalysed degradation. This optimised, improved and robust method will enable identification of keratinases ideally suited for application in the valorisation of the α-keratin found in natural wool fibres.