Serial blood draws for the assessment of trauma patients' hemoglobin (sHgb) and hematocrit (sHct) is standard practice. A device that would allow for continuous real-time, non-invasive monitoring of hemoglobin and tissue perfusion would potentially improve recognition, monitoring and resuscitation of blood loss. We developed a device utilizing diffuse optical spectroscopy (DOS) technology that simultaneously measures tissue scattering and near-infrared (NIR) absorption to obtain non-invasive measurements of oxy- (Hb-O(2)), deoxyhemoglobin (Hb-R) concentrations and tissue hemoglobin concentration (THC) in an animal model of hypovolemic shock induced by successive blood withdrawals. Intubated New Zealand White rabbits (N = 16) were hemorrhaged via a femoral arterial line every 20 min until a 20% blood loss (10-15 cc kg(-1)) was achieved to attain hypovolemia. A broadband DOS probe placed on the inner thigh was used to measure muscle concentrations of Hb-O(2) and Hb-R, during blood withdrawal. THC and tissue hemoglobin saturation (S(T)O(2)) were calculated from DOS [Hb-O(2)] and [Hb-R]. Broadband DOS-measured values were compared against traditional invasive measurements: systemic sHgb, arterial oxygen saturation (S(a)O(2)) and venous oxygen saturation (S(v)O(2)) drawn from arterial and central venous blood. DOS and traditional invasive measurements versus blood loss were closely correlated (r(2) = 0.96) showing a decline with removal of blood. S(T)O(2) and [Hb-O(2)] followed similar trends with hemorrhage, while [Hb-R] remained relatively constant. These measurements may be limited to some extent by the inability to distinguish between hemoglobin and myoglobin contributions to DOS signals in tissue at this time. Broadband DOS provides a potential platform for reliable non-invasive measurements of tissue oxygenated and deoxygenated hemoglobin and may accurately reflect the degree of systemic hypovolemia and compromised tissue perfusion.