Background: There is little data regarding the effect of tooth whitening on microhardness of white spot lesions. This study was conducted to investigate the effect of home-bleaching and laser-assisted in-office bleaching on microhardness of demineralized enamel. Material and Methods: Forty bovine incisors were selected and immersed in a demineralizing solution for 12 weeks to induce white spot lesions. Enamel blocks were prepared and randomly assigned to two groups of 20 each. The first group underwent home bleaching with 15% carbamide peroxide which was applied for 8 hours a day over a period of 15 days. In the second group, in-office bleaching was performed by 40% hydrogen peroxide and powered by irradiation from an 810 nm gallium-aluminum-arsenide (GaAlAs) diode laser (CW, 2W). This process was performed for 3 sessions every seven days, in 15 days. The specimens were stored in Fusayama Meyer artificial saliva during the experiment. Surface microhardness was assessed before and after the bleaching therapies in both groups. Results: Microhardness decreased significantly following both home bleaching and laser-assisted in-office bleaching ( p <0.05). There were no significant differences in hardness values among the two groups either before ( p =0.131) or after ( p =0.182) the bleaching procedures. Conclusions: Tooth whitening through home bleaching or laser-assisted in-office bleaching can result in a significant reduction in microhardness of white spot lesions. Therefore, it is suggested to take protective measures on bleached demineralized enamel.