This study investigates the efficacy of proteomic analysis of human remains to identify active Mycobacterium leprae infections in the past. Mycobacterial diseases, like leprosy, have plagued human populations for millennia. Thanks to effective treatment options, leprosy is not as widespread and deadly as in the past, yet remains endemic in certain regions with increasing concerns of strains becoming resistant to antibiotic treatments. We present a dual-enzyme, optimised extraction protocol, using trypsin and ProAlanase, to increase the recovery of non-collagenous proteins through a study of five individuals from a Mediaeval leprosarium cemetery, as well as four from a non-leprosy associated cemetery. Here we show that skeletal samples from the leprosarium individuals contain numerous immune proteins associated with modern leprosy, while those from a non-leprosy associated cemetery do not. Through this study, we advance a palaeoimmunology methodology and provide insights into the health of archaeological individuals and offer a means to triage samples for aDNA analysis.