A device and method utilizes a broadband diffuse optical spectroscopy (DOS) system to dynamically calculate the concentrations of multiple chromophores in vivo using a non-invasive probe. The device and method permit dynamic monitoring of multiple in vivo tissue chromophores non-invasively with sensitivities necessary for effective therapeutic monitoring. The device includes a probe containing first and second source optical fibers as well as first and second detector optical fibers. The probe is placed adjacent to a sample of interest and detects reflected light which is passed to a proximally located detector and spectrometer. The concentrations of multiple chromophores are determined in real time. In a preferred embodiment, the multiple tissue chromophores include at least two of methemoglobin (MetHb), deoxyhemoglobin (Hb-R), oxyhemoglobin (Hb-O2), water (H2O), and methylene blue (MB). The device and method can be used quantify and monitor methemoglobin formation in subjects suffering from methemoglobinemia.