Abstract Objective Assess color alteration and hydrogen peroxide (HP) penetration in human and bovine teeth using various in‐office bleaching protocols with different application times. Materials and Method s Thirty healthy human premolars and 30 healthy bovine incisors were divided into five groups and subjected to different bleaching protocols: 2 × 15 min, 1 × 30 min, 2 × 20 min, or 1 × 40 min, with a control group for each tooth type. All teeth were treated with 35% HP gel. Color alteration was measured using digital spectrophotometry before and 1 week after bleaching. HP concentration within the pulp was determined via UV–Vis spectrophotometry. Statistical analysis included one‐way ANOVA, Tukey's, and Dunnett's tests ( α = 0.05). Results All groups exhibited significant color alteration, with no statistically differences among them ( p > 0.05). However, significant differences were observed when compared with their respective control groups ( p < 0.05). HP penetration into the pulp was evident in all bleached teeth compared to the control groups ( p < 0.05), with the 2 × 20 group showing the highest HP levels within the pulp cavity, irrespective of tooth type ( p < 0.05). Conclusion A simplified 1 × 30‐min protocol can be recommended as it effectively maintains color alteration and HP penetration, irrespective of whether human or bovine teeth. Clinical Significance This study suggests that an in‐office dental bleaching protocol using a 1 × 30‐min session is recommended, as it ensures both effective color change and no increase in the amount of HP penetration.