Objective: Carious tissue discrimination in clinical operative caries management relies traditionally on the subjective hardness of carious dentine. Biochemical alterations within the lesion have the potential to discriminate the lesion zones objectively. This study aimed to determine the correlation between the biochemical proportions of amide I and phosphate moieties as these are the most prominent peaks found in dentine with the Knoop microhardness of carious dentine zones, using non-contact Raman spectroscopy. The null hypothesis investigated was that there was no correlation between Raman peak ratios, amide I: phosphateν1, and the Knoop microhardness within specific zones of a carious lesion. Methods: 423 scan points from 20 carious dentine lesion samples examined using high-resolution Raman spectroscopy. The peak ratio of the characteristic vibration mode of amide I (1650 cm −1) and phosphate (960 cm −1) bands were calculated, following a straight line path through the lesion to the pulp and correlated to corresponding Knoop microhardness measurements. Results: Using logistic regression analysis, clear correlations were found between the Knoop microhardness and Raman peak ratio cut-off values between caries-infected and caries-affected dentine (81.5 % sensitivity / 92.7 % specificity), with a lower specificity (2.7 %) found between caries-affected and sound dentine. Conclusion: This study concluded that non-contact Raman spectroscopy can be used in vitro to discriminate objectively between the different zones of a carious dentine lesion at high resolution, using the Raman peak ratios, amide I : phosphate ν 1. Clinical Significance: Specific biochemical alterations have the potential to be used in-vitro and in-vivo to identify the end-point of selective carious lesion excavation.