Prions are unique infectious agents which have been shown to be transmitted iatrogenically through contaminated surfaces. Surface contamination is a concern on reusable medical devices and various industrial surfaces, but there is currently no standard, accepted model to evaluate surface prion decontamination. In this report, a set of both in vitro and in vivo methods were investigated based on the contamination of surface through artificial exposure to infected brain. An in vitro surface contamination protocol was developed with subsequent biochemical detection of the prion protein (PrPres). In parallel, the in vivo investigations included the contamination of different types of surface materials (stainless steel or plastic wires) with different prion strains (scrapie strain adapted to hamsters 263K or bovine spongiform encephalopathy strain adapted to mouse 6PB1). The in vivo models with various prion strains and brain homogenate dilutions reproducibly transmitted the disease and a relationship was established between the infectivity titre, the transmission rate and the incubation period. Moreover, the in vivo models were studied for their ability to demonstrate the efficacy of heat and chemical-based decontamination methods, with similar results. The in vivo scrapie method described is proposed as a standard to evaluate existing and developing prion decontamination technologies.