The enamel erosion induced by acidic soft drinks is an increasingly important problem. The aim of this study was to investigate the effect of soft drinks on enamel erosion and the protection offered by representative modern toothpastes using a new 'in situ scanning electron microscopy (SEM)-replica technique'. Six patients were selected to receive in vivo enamel replicas, fabricated with a polyvinyl-siloxane/polyether impression material. Scanning electron microscopy was used to evaluate the morphology of enamel surface before and after exposure to lemon juice and SPRITE. The protective effectiveness of toothpaste was further evaluated with the same method. Furthermore, to validate the effectiveness of the in situ SEM-replica technique, we compared it to a direct in vitro SEM investigation on extracted teeth. Scanning electron microscopy investigation of the in situ replicas showed severe enamel morphology alterations after acidic soft drink exposure. On the other hand, it was also showed the protective effectiveness of toothpaste in preventing enamel erosion induced by acidic soft drinks. The direct in vitro SEM investigation provided similar enamel erosion results and proved the effectiveness of the in situ SEM-replica technique. Acidic soft drinks induce enamel erosion but regular use of toothpaste might reduce the risk for enamel erosion. The in situ SEM-replica technique provides an accurate method for tracing enamel morphology alterations and erosion induced by acidic soft drinks.