The aim of this study was to assess inter- and intra-examiner reproducibility and accuracy in the detection and assessment of occlusal caries in extracted human teeth using the newly developed fluorescence based camera VistaProof. Serial sectioning and microscopy are considered the gold standard. The occlusal surfaces of 53 teeth (99 investigation sites) were examined by two examiners with different levels of experience in cariology (one experienced dentist, one final-year dental student) and the VistaProof. Thereafter, the teeth were serially sectioned and assessed for lesion depth. The intraclass correlation coefficients for inter- and intra-examiner reproducibility for the fluorescence-based examinations were 0.76-0.95. There was a significant correlation between the fluorescence and histological examinations for both examiners (r (s) = 0.47 and 0.55, P < 0.01). At the D₁ diagnostic threshold (enamel and dentin lesions), sensitivity was 0.71-0.86 and specificity was between 0.32 and 0.76 at different cutoff values. At the D₃ diagnostic threshold (dentin lesions), sensitivity was 0.04-0.91 and specificity was 0.56-0.99 for both examiners. When the areas under the receiver-operating characteristic curves were compared, there was no significant difference in the performance between the examiners (P = 0.52 at the D₁ threshold, P = 0.81 at the D₃ threshold). The VistaProof demonstrated high reproducibility and good diagnostic performance for the detection of occlusal caries at various stages of the disease process. Novice and experienced examiners were able to apply this system as a supportive device for caries diagnostic and monitoring purposes. Sensitivity and specificity values varied depending on the cutoff values.