This study aimed to quantify the penetration of hydrogen peroxide, color change evaluation, surface morphology, and composition after application of desensitizing agents before in-office bleaching. Fifty premolars were sectioned, an acetate buffer was placed in the pulp chamber and divided into five groups (n=10). In the positive control group, only the in-office bleaching gel was used, and in the negative control group, no treatment was used. Three different desensitizing agents were applied: Desensibilize KF2%® group; Mi Paste® group, and Desensibilize Nano-P® group. The bleaching procedure was carried out with 35% HP. The absorbance of the resulting solution was determined in a spectrophotometer. Color change was assessed by using a digital spectrophotometer. Four additional premolars were assigned to the same groups above for analysis under scanning electron microscope, as well as to evaluate the elemental composition with X-ray dispersive energy spectrometry. Data were subjected to ANOVA and Tukey's test (α=0.05). All products reduced the penetration of HP in the pulp chamber. Mi Paste and Nano P were the products that yielded the lowest HP penetration, which was similar to the negative control group (p<0.001). No significant difference was detected in color change (p<0.001). Concerning enamel morphology, the groups that were analyzed after bleaching were observed a greater deposition of desensitizing agents on the surface. The use of desensitizing agents before tooth bleaching seems to be an alternative to reduce adverse effects of the tooth.