The aim of this in vitro study was to evaluate the performance of various contemporary detection tools at incipient occlusal caries. Thirty eight freshly extracted posterior teeth with non-cavitated occlusal caries were subjected to clinical examination and coding according to ICDAS criteria (n = 38). Standardized fluorescence images were taken with VistaProof (Dürr Dental, Bietigheim-Bissingen, Germany). Furthermore, the teeth were calculated with a laser fluorescence pen device (DIAGNOdent pen, Kavo, Biberach, Germany). In all detection methods, assessment of caries was performed twice (with 1-week interval) by two calibrated examiners. Finally, the caries lesions were validated in tooth sections by histological examination. The intra-examiner and inter-examiner kappa coefficient, sensitivity, and specificity were determined for all detection methods at enamel lesions (D1 threshold). Degrees of agreement of each method with the histological status were calculated using receiver operating characteristic statistics and the area under curve (Az values). The kappa intra-examiner/inter-examiner coefficient values (mean ± SD) were 0.74 ± 0.04/0.73 ± 0.07, 0.87 ± 0.04/0.82 ± 0.07, and 0.91 ± 0.06/0.83 ± 0.08 for clinical examination, DIAGNOdent pen, and VistaProof, respectively. The sensitivity for ICDAS was 0.80-0.86, for DIAGNOdent pen was 0.66-0.75, and for the VistaProof device was 0.97. The specificity for all the detection methods were 0.5 (0.02-0.99). The accuracy value for ICDAS was 0.76-0.81, for DIAGNOdent pen 0.66-0.71, and for the VistaProof device 0.92-0.95. The Az values (mean ± SD) were 0.431 ± 0.187, 0.583 ± 0.215, and 0.486 ± 0.207 for ICDAS, DIAGNOdent pen, and VistaProof examination, respectively. No significant differences in Az values were noted among the methods. All detection methods were presented with high inter-examiner and intra-examiner agreement. The new VistaProof device showed the best sensitivity, while DIAGNOdent pen demonstrated the worst one. Specificities were the same for all detection methods. Moreover, they presented the same performance in detection of incipient occlusal caries.