The aim of this in vitro study was to assess the effect of whitening gels of 35% carbamide peroxide (CP) on severity of dental caries, in case a caries lesion develops in future. Twenty samples prepared using healthy permanent bovine teeth were assigned to each of the following groups: (1) no tooth whitening (control group), (2) 15 min of whitening (experimental group 1), (3) 30 min of whitening (experimental group 2), and (4) 60 min of whitening (experimental group 3). An over-the-counter whitening agent containing 35% CP, potassium nitrate, and 0.11% fluoride ion (Opalescence PF Min 35%, Ultradent products Inc., Germany) was used. Vickers hardness number (VHN) was obtained for tooth surface using a Vickers hardness tester, and fluorescence loss (∆F %) was examined using quantitative light-induced fluorescence (QLF). Differences in the means among the groups were analyzed using one-way ANOVA and Tukey HSD post-hoc test. After whitening treatment, there were no statistically significant differences in VHN and ∆F between the control group and experimental groups (p > 0.05). Therefore, application of fluoride-containing 35% CP tooth whitening gel for a short duration of 60 min or less was found to have no impact on the severity of initial dental caries.