Infectious prions readily adhere to common surfaces, retain infectivity, and are highly resistant to conventional decontamination, posing significant biosafety challenges in the medical and research environments. Recent occupational exposures underscore the urgency of improving safety measures. Here, we describe an approach combining foam-swab surface sampling and protein misfolding cyclic amplification to enhance prion surveillance. Our results demonstrate the ability to detect prions most relevant to human health directly from contaminated surfaces, even at 100 million-fold dilutions of the brain. We applied our method to assess the completeness of prion decontamination and show that high prion quantities can resist even approved inactivation methods. Finally, we applied our method in 2 real-world scenarios including the decommissioning and repurposing of a prion research facility and the active surveillance of residual prion contamination in an operational laboratory. Our methodology offers a robust and efficient tool for detecting residual prion contamination, enhancing laboratory safety. Recent reports have documented prion infections during occupational exposure in laboratory research. We implemented a methodology for prion surveillance in the laboratory, which can be used to monitor prion contamination, assess the efficiency of decontamination, and repurpose equipment and rooms.