The aim of this study is to assess the extent of early carious lesions using the International Caries Detection and Assessment System (ICDAS) II and DIAGNOdent pen compared with microCT examination. Forty-eight molars and premolars were qualified for the study. The visual and the DIAGNOdent pen device examinations were carried out by two examiners. The actual extent of the lesions was determined using micro-CT examination. The optimal cutoff test value was determined using the Youden index. For the two methods, inter- and intra-examiner reproducibility was calculated using intraclass correlation coefficient. The sensitivity, specificity, and accuracy of these methods were compared using the mid-P McNemar test. The level of significance was set at p < 0.05. In terms of the enamel threshold for the ICDAS II scale, insignificantly higher mean evaluated parameters were noticed in comparison with the DIAGNOdent pen device. For the dentin threshold, all the assessed parameters had higher mean values on the ICDAS II scale compared with the DIAGNOdent pen. The optimal cutoff points of the enamel and dentin thresholds are lower than that suggested by the manufacturer. The ICDAS II and the DIAGNOdent pen device were characterized by high inter - and intra-observer reproducibility of the test results. To improve the diagnostic efficiency of the DIAGNOdent pen, modifying the cutoff values recommended by the manufacturers should be considered. After modifying the cutoff values, the combination ICDAS II and DIAGNOdent pen device would be the better choice in order to detect caries on occlusal surfaces.