Objective:To analyze color change, microhardness and chemical composition of enamel bleached with in-office bleaching agent with different desensitizing application protocols.Materials and Methods:One hundred and seventeen polished anterior human enamel surfaces were obtained and randomly divided into nine groups (n = 13). After recording initial color, microhardness and chemical composition, the bleaching treatments were performed as G1: Signal Professional White Now POWDER&LIQUID FAST 38% Hydrogen peroxide(S); G2: S+Flor Opal/0.5% fluoride ion(F); G3: S+GC Tooth Mousse/Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) paste(TM); G4: S+UltraEZ/3% potassium nitrate&0.11% fluoride(U); G5: S+Signal Professional SENSITIVE PHASE 1/30% Nano-Hydroxyapatite (n-HAP) suspension(SP); G6: S-F mixture; G7: S-TM mixture; G8: S-U mixture; G9: S-SP mixture. Color, microhardness and chemical composition measurements were repeated after 1 and 14 days. The percentage of microhardness loss (PML) was calculated 1 and 14 days after bleaching. Data were analyzed with ANOVA, Welch ANOVA, Tukey and Dunnett T3 tests (p<0.05). Results: Color change was observed in all groups. The highest Delta E was observed at G7 after 1 day, and Delta E at G8 was the highest after 14 days (p<0.05). A decrease in microhardness was observed in all groups except G6 and G7 after 1 day. The microhardness of all groups increased after 14 days in comparison with 1 day after bleaching (p>0.05). PML was observed in all groups except G6 and G7 after bleaching and none of the groups showed PML after 14 days. No significant changes were observed after bleaching at Ca and P levels and Ca/P ratios at 1 or 14 days after bleaching (p>0.05). F mass increased only in G2 and G6, 1 day after bleaching (p<0.05). Conclusions: The use of desensitizing agents containing fluoride, CPP-ACP, potassium nitrate or n-HAP after in-office bleaching or mixed in bleaching agent did not inhibit the bleaching effect. However, they all recovered microhardness of enamel 14 days after in-office bleaching.