The ability to quantify the contributions of hemoglobin (Hb) and myoglobin (Mb) to in vivo optical spectra has many applications for clinical and research use such as noninvasive measurement of local tissue O(2) uptake rates and regional blood content. Recent work has demonstrated an approach to independently measure oxygen saturations of Hb and Mb in optical spectra collected in vivo. However, the utility of this approach is limited without information on tissue concentrations of these species. Here we describe a strategy to quantify the contributions of Hb and Mb to in vivo optical spectra. We have found that the peak position of the deoxy-heme peak around 760 nm in the optical spectra of the deoxygenated tissue is a linear function of the relative contributions of Hb and Mb to the optical spectra. Therefore, analysis of this peak position, hereafter referred to as wavelength shift analysis, reveals the relative concentration of Hb to Mb in solutions and intact tissue. Biochemical analysis of muscle homogenates confirmed that the wavelength shift of the combined Hb/Mb peak in in vivo spectra reflects the ratio of concentrations (Hb/Mb) in muscle. The importance of quantifying the Hb contribution is illustrated by our data demonstrating that Hb accounts for approximately 80% of the optical signal in mouse skeletal muscle but only approximately 20% in human skeletal muscle. This advance will facilitate comparison of the metabolic properties between individual muscles and provides a fully noninvasive approach to measuring local respiration that can be adapted for clinical use.