This study aimed to assess swept source optical coherence tomography (SS-OCT) for in vitro and in vivo detection of enamel white spot lesion (WSL). WSLs without surface breakdown on 33 extracted human posterior teeth were non-invasively scanned using SSOCT. The teeth were then cross-sectioned and imaged under confocal laser scanning microscope (CLSM) and light microscopy (LM). SS-OCT cross-sectional images were compared with CLSM and LM. WSL shapes in SS-OCT images closely corresponded to those of LM. There were significant correlations (p<0.001) in WSLs depth between SS-OCT and LM (r=0.92), SS-OCT and CLSM (r=0.80) and CLSM and LM (r=0.85). Six WSLs were also evaluated clinically using SS-OCT; clear in-depth images of these natural WSLs were obtained in vivo. SS-OCT appears to be an effective tool for observation of the internal structure of WSLs, enabling quantitative assessment of WSL depth. Such data can be considered in the clinical management of WSLs.