There is an increasing need for effective biomonitoring tools that quantify patterns of habitat occupancy by reptile species. Environmental DNA (eDNA) has been regarded as an emerging tool to detect specific target species; however, the dynamics of accumulation and degradation of eDNA in terrestrial environments are poorly understood. This study determines the time required for terrestrial snakes to leave enough eDNA behind to become detectable (accumulation time) as well as its persistence (degradation time). By targeting mitochondrial cytochrome oxidase subunit I and 12S rRNA genes of Red Cornsnakes (Pantherophis guttatus) in a controlled laboratory setting, we found that eDNA can be detected 3.5 h after the snakes had contact with soil and for up to 6 d after their removal. Estimated accumulation rate of Pantherophis guttatus eDNA per gram of snake biomass per hour was 12.6 μg. We also evaluated the applicability of eDNA detection under field conditions by targeting the mitochondrial cytochrome b gene of a cryptic invasive species in South Florida, Burmese Pythons (Python bivittatus). Soil samples were derived from two groups of field sites: telemetry-monitored refugia (i.e., radiotelemetry evidence of python presence) and telemetry-absent refugia (i.e., no telemetry evidence, but monitored with a burrow camera at time of sample collection). We were able to detect the presence of python eDNA in 66.7% of the telemetry-monitored sites that fit within our laboratory-defined residence and degradation time window. Additionally, at the telemetry-absent sites, no eDNA from Burmese Pythons was detected and burrow cameras did not detect their presence. We concluded that eDNA technology using soil can be an effective detection tool for terrestrial snakes, particularly when used with other traditional tracking and sampling methods.