Methemoglobin concentration is an important pathophysiological biomarker, reflecting the oxygen-carrying and oxygen-releasing capabilities of hemoglobin (Hb). Raman spectroscopy is used to develop a novel technique for determining the methemoglobin concentration. Raman activity combined with two-dimensional correlation analysis is an attractive method for investigating Hb oxidation, exhibiting several relevant peaks in the range of 1200-1650 cm(-1). Methemoglobin concentration is estimated by measuring the intensity of Raman peaks in the ranges of 1210-1230 cm(-1) and 1340-1380 cm(-1) with 785-nm excitation. The correlation between Raman-based methemoglobin concentration estimations and the methemoglobin concentration measured using spectrophotometry was highly significant. These results suggest the potential of Raman spectroscopy as a new quantitative approach to determine the methemoglobin concentration.