Objectives: To measure the level of hydrogen peroxide penetration into the pulp chamber and to evaluate its whitening efficacy when the tooth is treated with an innovative double layer of potassium nitrate desensitizing agent and hydrogen peroxide whitening gel. Methods: Extracted human molar teeth (n=160) were collected. Roots were trimmed 3 mm apical to the cemento-enamel junction, the pulp was removed and a cavity prepared, leaving a standardized wall of 2 mm thickness, which retained 100 µL of acetate buffer solution. A standardized whitening area of 6 mm in diameter was established buccally by painting the rest of the tooth with gray nail varnish. The teeth were randomly assigned into four groups. Group 1: no treatment (glycerin gel, negative control); Group 2: double layer of 20 L 5% potassium nitrate (Relief ACP, Philips Oral Healthcare) and 100 L 25% hydrogen peroxide (Zoom Chairside Whitening Gel, Philips Oral Healthcare); Group 3: double layer of 40 L 5% potassium nitrate and 100 L 25% hydrogen peroxide; and Group 4: 100 L 25% hydrogen peroxide (positive control). All groups received three 45-minute sessions of in-office whitening with light activation at 3-day intervals. Hydrogen peroxide penetration was assessed spectrophotometrically using leucocrystal violet and horseradish peroxidase. Color measurements were assessed with VITA Easyshade at baseline, 1-day, and 1-month post-whitening. Tooth color change was measured per Commission Internationale de l'Eclairage methodology. One-way ANOVA with Tukey multiple comparisons adjustment was performed to compare group differences in overall tooth color change (Delta E*) and hydrogen peroxide penetration (type I error = 0.05). Results: Hydrogen peroxide penetration levels were not significantly different between Group 2 and Group 4; however, the levels were significantly higher than Group 1 and Group 3 (p< 0.0001). Groups 2, 3 and 4 showed no differences for overall tooth color change but differed significantly from the negative control group (Group 1) at 1-day and 1-month post-whitening. Conclusions: Hydrogen peroxide penetration was affected when pretreated by potassium nitrate desensitizer; however, the double layer technique did not adversely affect the whitening efficacy.