Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.