Assess color alteration and hydrogen peroxide (HP) penetration in human and bovine teeth using various in-office bleaching protocols with different application times. 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). 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). 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. 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.