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A precise evaluation of caries excavation endpoint is essential in clinical and laboratory investigations. Caries invasion differentiates dentin into structurally altered layers. This study assessed these changes using Raman spectroscopy and Vickers microhardness. Ten permanent molars with occlusal and proximal carious lesions were assessed and compared at 130 points utilizing four Raman spectroscopic peaks: phosphate v1 at 960 cm -1 , amide I (1650 cm -1 ), amide III (1235 cm -1 ) and the C-H bond of the pyrrolidine ring (1450 cm -1 ). The phosphate-to-amide I peak ratio and collagen integrity peak ratio (amide III: C-H bond) of carious zones were calculated and compared in both lesions. The former ratio was correlated to 130 Vickers microhardness indentations through lesions. The caries-infected dentin (CID) exhibited low phosphate peak, but higher amide I, III and C-H bond peaks than other zones in both lesions. The peaks in amide regions (I and III) varied in occlusal versus proximal lesions. A high correlation was found between mineral: matrix peak ratio and equivalent microhardness number within carious lesions, while the collagen integrity peak ratio was applied in proximal lesions only. Raman spectroscopy detected changes in the mineral and matrix contents within different carious zones and regions.