Environmental DNA (eDNA) is DNA shed by organisms into the soil or water with which they interact. It offers an opportunity to survey wildlife non-invasively, which is especially useful for endangered and threatened species. The Eastern Indigo snake (Drymarchon couperi) is a Federally threatened snake species with historic habitat in the dry forests of the southeast United States. We developed a novel protocol for Indigo snake presence detection using eDNA from soil samples. These soil samples were obtained from outdoor snake enclosures from the Orianne Center for Indigo Conservation in Eustis, FL and from a captive snake release site in Conecuh National Forest in Alabama. Ten soil samples from areas with confirmed snake activity were used to test eDNA extraction from soil using Longmire’s buffer and the Zymo Quick-DNA Miniprep Plus Kit. We successfully detected snake presence with DNA from genes NADH dehydrogenase 4 (ND4) and cytochrome b (CYTB). This method was then applied to the field at Archbold Biological Station, showing that this protocol can be used as an inexpensive, simple, non-invasive technique of surveying the threatened indigo snake. The broader implication of this novel methodology is its application to other elusive endangered and threatened species with which presence can be obtained from eDNA soil samples.