Enamel defects (EDs) are qualitative and/or quantitative disturbances of the dental surface. To date, the responsiveness to remineralizing treatments has been studied ex vivo, on dental sections from extracted teeth. The present research aims to establish if in vivo reflectance confocal laser scanning microscopy is able to visualize the changes in the enamel architecture on living teeth, before, during and after remineralizing treatments by casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). As proof-of-concept study, 17 consecutive children affected by EDs were enrolled and 38 EDs were considered. A CPP-ACP mousse was applied twice a week for 6 weeks and clinical and microscopic images were collected before, during and after the treatment for evaluating the changes occurred. For in vivo microscopic imaging, a reflectance confocal laser scanning microscope (RCM) for in vivo use was adopted. In this study RCM was proven to be able to visualize in vivo and at microscopic resolution the changes occurred during the remineralizing processes without needing for dental extractions and histopathological procedures. This in vivo RCM capability could encourage its clinical application in monitoring responsiveness to enamel therapies.