This in vitro study aimed to compare outcomes of dental caries detection using visual inspection classified according to the International Caries Detection and Assessment System (ICDAS) with objective assessments using a well-established laser fluorescence system (Diagnodent pen) and a novel diffuse reflectance spectroscopy (DRS) device. One hundred extracted permanent premolars and molars were utilized, including sound teeth, teeth with non-cavitated caries, or teeth with small cavitated lesions. A total of 300 regions of interest (ROIs) were assessed using each detection method. Visual inspection, being a subjective method, was performed by two independent examiners. The presence and extent of caries were histologically verified according to Downer's criteria, serving as a reference for other detection methods. Histological results revealed 180 sound ROIs and 120 carious ROIs, categorized into three different extents of caries. Overall, there was no significant difference between the detection methods in sensitivity (0.90-0.93) and false negative rate (0.05-0.07). However, DRS exhibited superior performance in specificity (0.98), accuracy (0.95), and false positive rate (0.04) compared to other detection methods. Although the tested DRS prototype device exhibited limited penetration depth, it shows promise as a method, particularly for the detection of incipient caries.