Environmental DNA (eDNA) sampling has become an essential and sensitive detection method to monitor native and invasive species populations. In Europe, some crayfish are invasive and established populations can spread rapidly in freshwater environments. Tracking their presence (early detection) and distribution is difficult using live captures especially since setting traps is more time consuming and less cost effective. Therefore, the potential of eDNA assay as a monitoring tool is explored. Fifty locations in Brussels (Belgium) were sampled and tested for the presence of four invasive crayfish species (Procambarus clarkii, Procambarus virginalis, Faxonius limosus and Pontastacus leptodactylus). The present investigation aims at i) testing existing qPCR protocols and evaluating their suitability at detecting the targeted invasive crayfishes, ii) evaluating the eDNA assays reliability by comparing the results against live capture data iii) evaluating the advantages and disadvantages of eDNA assays vs live captures. Different qPCR protocols were tested against selectivity and sensitivity and two different filter protocols were applied on the field. From seven existing protocols four qPCR assays were selected to detect the presence of the targeted invasive crayfishes. At least three invasive species have been confirmed for ten locations in Brussels using eDNA. The outcome of this study will influence the strategy of future monitoring efforts.