ObjectivesTo evaluate the penetration of hydrogen peroxide into the pulp chamber, color change, and enamel morphology of human teeth with different substrates (sound and white spot), subjected to the application of a resin infiltration (RI) and in-office bleaching at different time intervals (immediately and 7 days after RI application). Materials and methodsFifty-four sound human premolars were randomly distributed into six groups (n = 9), all subdivided according to substrate (sound and white spot): bleaching with 35% hydrogen peroxide only (HP); RI followed by immediate bleaching (RI/HPi); and RI followed by bleaching after 7 days (RI/HP7d). The concentration of hydrogen peroxide (mu g/mL) was measured by UV-Vis spectrophotometry, color change was evaluated using a digital spectrophotometer (Delta E00 and WID), and enamel surface morphology was analyzed by scanning electron microscopy (SEM) (alpha = 0.05). ResultsIn the groups without RI (HP), teeth with white spot lesions showed approximately 40% greater hydrogen peroxide penetration than sound teeth (p = 0.15). RI significantly reduced this penetration, regardless of the substrate or bleaching time (p > 0.02). According to WID values, the groups treated with RI (RI/HPi and RI/HP7d) achieved a final color similar to that of sound enamel treated with 35% HP. Enamel morphology was more homogeneous in the groups that received resin infiltration. ConclusionsThe application of RI prior to in-office bleaching significantly reduced hydrogen peroxide penetration into the pulp chamber without compromising bleaching efficacy. In teeth with white spot lesions, it led to a final shade comparable to that of sound teeth and resulted in a more uniform enamel morphology. Clinical relevanceResin infiltration is an effective, minimally invasive approach for treating white spot lesions, improving bleaching outcomes and limiting hydrogen peroxide penetration.