Background Brazil is home to a multitude of venomous snakes, perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of a photo of the snake or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. Results In this paper, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp. specific antibody sandwich pairs. The sandwich pairs were used to develop a prototype LFA that was able to detect venom from several different Bothrops spp. The LFA had a limit of detection (LoD) of 0.5 ng/mL in urine, when read with a commercial reader, and a visual LoD of approximately 25 ng/mL. Significance The work presented here serves as a proof of concept of a genus-specific venom detection kit, which could support physicians in diagnosing Bothrops envenomings. Although further optimization and testing is needed before the LFA can find clinical use, such a device could aid in decentralising antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.