Red blood cell (RBC) transfusion guidelines are designed to maintain adequate tissue oxygenation by increasing blood oxygen-carrying capacity. However, since tissue oxygenation is not measured, RBC transfusion guidelines are mostly subjective. Clinical evidence of oxygen transport/consumption mismatches in infants is often unclear and confounded by multiple factors. Invasive hemoglobin measurements can contribute further to anemia if performed too frequently. Diffuse optical spectroscopy (DOS) is a noninvasive quantitative method to measure the tissue oxy, deoxy, and total hemoglobin concentrations (ctO2Hb, ctHb, ctTHb), as well as mixed arterial-venous tissue hemoglobin saturation (stO2). Our objective is to determine if DOS can assess changes in tissue oxygenation in very low birth weight (VLBW) infants undergoing RBC transfusions. DOS measurements of ctO2Hb and ctHb are performed on 10 VLBW infants before and within 24 h after RBC transfusion. Seven nontransfused infants are studied to evaluate hemodynamic variations independent of RBC transfusion. Tissue near-infrared absorption and scattering values are measured using a four-wavelength (690, 750, 810, and 830 nm) frequency-domain tissue oximeter (OxiplexTS, ISS, Champaign, Illinois). In transfused subjects, DOS demonstrates significant increases in ctO2Hb (48±13 versus 74±20 µM, p<0.002), ctTHb (87±17 versus 107±24 µM, p=0.004), and stO2 (54±8 versus 68±6%, p<0.004) post-transfusion. DOS measurements correlate with mean hemoglobin increases for all infants (r=0.83, p<0.0001). No significant DOS changes occurred in the nontransfused group. Calculations of the differential path length for these transfused subjects show high variability (~20%). DOS may serve as a noninvasive bedside tool to assess tissue oxygenation in infants and provide a functionally based transfusion trigger.