Background: This study investigated the effect of using different agents for protecting enamel proximal surfaces against acidic attack after interproximal reduction (IPR) using the trans micro radiography technique. Methods: Seventy-five sound-proximal surfaces were obtained from extracted premolars for orthodontic reasons. All teeth were measured miso-distally and mounted before being stripped. The proximal surfaces of all teeth were hand stripped with single-sided diamond strips (OrthoTechnology, West Columbia, SC, USA) followed by polishing via Sof-Lex polishing strips (3M, Maplewood, MN, USA). Three-hundred micrometers of enamel thickness was reduced from each proximal surface. The teeth were randomly divided into 5 groups: group 1 (control un-demineralized) received no treatment, group 2 (control demineralized) had their surfaces demineralized after the IPR procedure, group 3 (fluoride) specimens were treated with fluoride gel (NUPRO, DENTSPLY, Charlotte, NC, USA) after the IPR, group 4 (Icon) resin infiltration material (Icon Proximal Mini Kit, DMG, Bielefeld, Germany) was applied after IPR, group 5 (MI varnish) specimens were treated with Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) containing varnish (MI Varnish, G.C, USA, St. Alsip, IL, USA) after the IPR. The specimens in (groups 2–5) were stored in a 4.5 pH demineralization solution for 4 days. The trans-micro-radiography (TMR) technique was conducted to evaluate the mineral loss (∆Z) and lesion depth of all specimens after the acid challenge. The obtained results were analyzed statistically using a one-way ANOVA at a significance level of α = 0.05. Results: The MI varnish recorded significant ∆Z and lesion depth values compared to the other groups p > 0.05. There was no significant difference in ∆Z and lesion depth between the control demineralized, Icon, and fluoride groups p < 0.05. Conclusion: The MI varnish increased the enamel resistance to acidic attack, and thus can be considered an agent capable of protecting the proximal enamel surface after IPR.