Objective: This study aimed to evaluate the effectiveness of different mechanical methods for the removal of demineralized dentin. Methods: Healthy human third molars were prepared in such a way that the flat occlusal surfaces of the dentin were exposed and longitudinally sectioned in a vestibular-lingual direction. One section of each tooth was submitted to the pH-cycling model, while the other section was kept intact. The tooth sections were joined, and a single operator performed dentin removal using a steel bur, a hand instrument, or a polymer bur. The tooth's sections were then separated, and digital images were obtained. The depth of the prepared cavities and the microhardness measurements were checked and analyzed statistically using the Kruskal-Wallis One-Way ANOVA in Ranks, the Tukey test, while desmineralized dentin were compared using the One-Way ANOVA, and the Holm-Sidak method (p < 0.05). Results: The steel bur produced the deepest cavities in mineralized and demineralized dentin. The polymer bur generated the shallowest cavities in demineralized dentin. The measurements of microhardness of the deepest surfaces of the cavities prepared in demineralized dentin indicated that the steel bur and hand instrument presented similar values, while those values produced by polymer burs proved to be lower. Conclusion: The effectiveness of the removal of demineralized dentin varied among the three methods used in this study. The polymer bur proved to be the most conservative of the methods used. By contrast, the steel bur and hand instrument showed a similar effectiveness in the removal of dentin, according to the microhardness of the remaining dentin, even though they produced different cavity depths.