Invasive Central American boas (Boa imperator) have established a reproducing population on the western side of St. Croix, U.S. Virgin Islands but prevalence throughout the island is largely unknown. The large snakes threaten endemic and endangered species through competition and predation, jeopardizing island biodiversity. Environmental DNA (eDNA) methods were used to investigate occurrence and focal areas for management efforts in the Salt River Bay National Historical Park and Ecological Preserve (SARI). To validate a previously developed assay, we collected tissue samples and 13 × 60 mL water samples from a captive boa enclosure in St. Croix. We implemented this assay for both a pilot and main field sampling effort. The pilot in December 2023 resulted in analysis of 7 × 60 mL water samples per site from SARI (3 sites) and from the western forests where boas are established (1 site). The main sampling event in July 2024 collected 15 × 60 mL water samples per site within SARI (11 sites) and western forests (4 sites). Pilot sample replicates were filtered individually, while main samples were consolidated into groups, resulting in seven replicates for pilot sites and five replicates for main event sites, totaling 103 environmental samples. eDNA was isolated using a modified phenol-chloroform isolation method to remove PCR inhibitors, and target eDNA was amplified using droplet digital PCR technology. Water samples from the captive boa amplified target eDNA in 12 of 13 samples, indicating assay effectiveness ex-situ. Low concentrations of eDNA (below the 95% limit of detection) were amplified in 4 of 5 sites in the western forest and in 8 of 14 sites within the National Historic Park. Overall, boa eDNA concentrations were consistently low, as expected in water samples targeting a semi-arboreal snake species with a low rate of eDNA shedding. Further optimization of methods could enable recovery of greater eDNA concentrations in future studies. Additional eDNA method testing and ground-truthing may help to improve the assessment of invasive Boa imperator in St. Croix.