The aim of the study was to identify morphological changes in dentine and the relationship with hardness in endodontic retreatment using a rinse solution based on 10% citric acid.Method: This is an experimental in vitro study. 60 monoradicular premolar teeth were used and divided into 3 groups (20 teeth each) for testing three different retreatment protocols: Group 1: Mechanical retreatment with 5% Sodium Hypochlorite (NaClO), Group 2: Retreatment using Xylol and 5% NaClO, Group 3: Retreatment using citric acid and 5% NaClO; In addition, two control groups were used: Control group 1: Unprepared teeth (5 pieces), Control group 2: Unprepared teeth, irrigated without obturation (5 pieces).These 70 teeth were split longitudinal in two halves exposing the canal dentine for microhardness (Vickers) and Raman spectroscopy tests. Results: After endodontic retreatment Xylol-based a significant reduction in microhardness of radicular dentine was observed (ANOVA y pos-hoc Tukey statistical tests were performed). For all three retreatment procedures, traces of filling material were detected through Raman spectroscopy measurements. Xylol-based retreatment procedure showed higher rate of filling material than citric acid-based retreatment which exhibited the lowest. However, Raman spectroscopy is unable to prove phosphate and carbonate losses in dentine canals. Conclusions: Compared to Xylol desobturation treatment, the use of 10% citric acid results in lower reduction of radicular dentine microhardness, lower amount of obturation material residuals and non-observable demineralizing effects.