Detection of vulnerable plaques as the underlying cause of myocardial infarction is at the center of attention in cardiology. We have previously shown that infiltration of inflammatory cells in atherosclerotic plaques renders these plaques relatively hot and acidic, with substantial plaque temperature and pH variation. The objective of this investigation was to determine whether near-infrared diffuse reflectance spectroscopy (NIRS) could be used to non-destructively measure the tissue pH in atherosclerotic plaques. NIRS and tissue pH electrode measurements were taken on freshly excised carotid plaques maintained under physiological conditions. The coefficient of determination between NIRS and the pH microelectrode measurement was 0.75 using 17 different areas. The estimated accuracy of the NIRS measurement was 0.09 pH units. These results demonstrate the feasibility of using NIRS tissue pH in freshly excised atherosclerotic plaques in light of marked pH heterogeneity and warrants future in-vivo investigations on pH measurement of atherosclerotic plaques.