Interproximal enamel reduction (IER) is a minimally invasive therapeutic procedure commonly used in orthodontics to address both functional and aesthetic issues. Its mechanical effects on enamel surfaces induce the formation of grooves, furrows, scratches, depressions, and valleys. The aim of this study was to assess the enamel surface roughness resulting after the application of currently available methods for interproximal reduction. Ninety freshly extracted human teeth were divided into six groups and subjected to the stripping procedure, using a different method for each group (diamond burs, abrasive strips of 90 μm, 60 μm, 40 μm, 15 μm, and abrasive discs). A single individual performed stripping according to the manufacturer’s recommendations, involving interproximal reduction on one tooth’s proximal face and leaving the other side untreated. Qualitative and quantitative assessment of the enamel surfaces was carried out using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM), obtaining 2D and volumetric 3D images of the enamel surface microstructure and nanostructure. The study found that diamond burs and abrasive strips of 60 μm and 90 μm increased enamel roughness due to intense de-structuring effects, while the 40 μm polisher had a gentler effect and 15 μm abrasive strips and polishing discs preserved enamel surface quality and removed natural wear traces.