The aim of this study was to evaluate the antimicrobial, stain and protein removal efficacy of denture or orthodontic appliance cleansers using in vitro test methods. Experimental cleansers were applied for experimental time in each evaluation method. To evaluate the microorganism removal efficacy, C. albicans and S. mutans removal rate was calculated from the specimen surface. Stain and protein removal rate was calculated using the spectrophotometer. Experimental cleansers significantly affected the microorganism removal rate for both C. albicans and S. mutans, as well as the stain and protein removal rates, at each experimental time (p<0.05). As the application time increased, the stain and protein removal rates of all experimental cleansers significantly increased (p<0.05). The present study provided in vitro evaluation methods to assess the efficacy of denture or orthodontic appliance cleansers. Also, manufacturers and researchers can predict clinical outcomes and ensure proper hygiene management of dentures or orthodontic appliances.