The stability of the infectious agent causing variant Creutzfeldt–Jakob disease (vCJD) has highlighted the importance of cleaning surgical instruments for controlling potential spread of iatrogenic CJD. In this study, thermostable adenylate kinases (tAKs) in test soil were coated on to stainless steel and these surrogate agents used to evaluate the efficacy of a range of cleaning chemistries in a bench-top washer disinfector (btWD), or as a pre-soak either with or without subsequent treatment by btWD. Two tAKs were tested initially for ease of removal, the most persistent being Sulfolobus acidocaldarius-derived tAK which was used for evaluating the cleaning chemistries. Conventional chemistries were generally more effective when used in a btWD than as pre-soaks. Cleaning efficacy improved when pre-soaks were followed by treatment with intermediate performing enzymes, demonstrating greater than additive effect on residual tAK activity. Three of the four prion-directed chemistries reduced residual tAK activity to below the limit of quantification (LoQ) by more than 4.8 log₁₀; <175pg tAK remaining as a pre-soak alone. A conventional alkaline cleaning product also reduced residual tAK activity to below the LoQ but only when used in a btWD. tAK soil dried on to the device was removed less efficiently than tAK soil still moist on the device, with a 320-fold and 28-fold increase in residual tAK activity for pre-soak and btWD, respectively. The study demonstrated the potential for a tAK indicator to describe the effectiveness of protein removal using different chemistries or treatment processes.