Innate immunity in arthropods is achieved largely through melanization which is in turn the result of the prophenoloxidase (ProPO) activation cascade; a series of biochemical reactions triggered by the immune identification of pathogen-recognition proteins (PRPs). Within this activation cascade, inactive proPO is cleaved to form the reactive enzyme phenoloxidase (PO). Methods of detecting PO are used to assess an arthropod's ability to respond to immune challenges. These detection assays have been described for some arthropods, especially those of commercial value, but none are available for Euastacus, a genus within the superfamily Parastacoidea. This study is the first step in developing a standardized protocol for the detection and quantification of PO activity in wild or captive Murray crayfish Euastacus armatus. Hemolymph extracts from 49 crayfish were assessed for PO activity using an assay measuring the conversion of L-dopa (3,4-dihydroxy-L-phenylalanine) into dopachrome. Short periods (up to 15 min) out of water did not cause any measurable change in PO activity. Phenoloxidase activity was detected in captive (n = 24, stressed) and wild (n = 25, healthy) crayfish with captive crayfish showing lower levels of PO possibly indicating immunosuppression. The proven protocol is the first of its kind to propose a standardized methodology for the detection and quantification of PO activity in Murray crayfish hemolymph as a means of determining stress.