Abstract: Non-indigenous species (NIS) reach every corner of the world, at times wreaking havoc on ecosystems and costing the global economy billions of dollars. Metabarcoding-based tools optimized for biogeographic regions enable rapid and accurate early detection across a wide taxonomic range to allow quick implementation of eradication or control efforts and potentially mitigate some of the devastating effects of NIS worldwide. Here, I will outline the steps and considerations for developing a regional NIS metabarcoding detection tool based on research conducted in British Columbia, Canada. We developed a multi-marker metabarcoding assay tailored to the native and non-indigenous species assemblages in the north-east Pacific Ocean. After undergoing field validation, we compared NIS biofouling communities detected from traditional settlement plates and eDNA, and found higher NIS diversity detected in eDNA. Substrate type (DNA from water and zooplankton samples) and seasonal fluctuations also influenced the total number of NIS detected from metabarcoding and should be an important consideration for monitoring studies. NIS detection using metabarcoding presents a unique set of molecular and field sampling challenges that need to be considered for a cost-effective, rapid, and accurate biosurveillance tool. Optimized molecular surveys are appropriate for broad scale detection of marine NIS due to their amenability to high sample numbers and throughput.