Carbamide peroxide (CP) is widely used as a tooth-whitening agent in self-administered tooth-bleaching products. In this study, the effects of 5% and 10% CP on dentinal collagen structure and chemical properties were evaluated in vitro. Thirty-five intact teeth were exposed to 2 whitening protocols (2 or 4 h daily) with either 5% or 10% CP gel for 1 wk. Shade changes before and after the whitening protocol were captured colorimetrically using a spectroshade. Collagen scaffold models and demineralized dentine disc samples were prepared and exposed to CP droplets (5% or 10%). Structural changes were investigated using electron microscopy. Finally, mineralized dentine disc samples were prepared postbleaching to assess chemical changes resulting from CP exposure in dentinal collagen using Raman spectroscopy. Results showed a difference in tooth shade when exposed to 5% and 10% CP whitening protocols, with a significantly ( P ≤ 0.01) greater change reported for the 10% CP/4-h group. Imaging of the collagen scaffold model following exposure to CP showed a gelatinization process indicating that the free radical by-products from CP are able to disrupt the quaternary structure of noncrosslinked collagen. The most significant damage on the collagen scaffold was seen for the 10% CP exposure for 4 h. Imaging of the demineralized discs displayed the same glassy amorphous layer appearance as found in the collagen scaffold. Raman spectra of the mineralized dentine discs showed a significant decrease ( P ≤ 0.01) in the integrated area of amide I and amide III values in the 4 test groups following CP application. Amide I was more affected as both the exposure time and concentration of CP increased. Despite the claimed safety of whitening agents, this in vitro study concludes that even low concentrations of CP result in a deleterious change in dentinal collagen.