The objective of this study was to investigate the correlation between CT depth, indentation depth determined by a new hardness tester (Cariotester), and the transverse microradiography (TMR) parameters, i.e., lesion depth and mineral loss. For that purpose, this study evaluated the feasibility of using Cariotester as a root caries diagnostic system and capability of Cariotester to detect effect of fluoride application on inhibiting dentin demineralization. Fluorides were applied to bovine root dentin specimens, which were subsequently demineralized for 1–21 days and then CT depth and TMR parameters were assessed. There were significant correlations between CT depth and TMR parameters in fluoride and non-fluoride groups. There were significant differences between fluoride and non-fluoride groups for CT depth and TMR parameters respectively. Current results suggested that Cariotester may be capable of providing an objective evaluation of root caries progression and the fluoride effect on inhibiting dentin demineralization.