The US is about 1.5 days away from exhausting its entire blood supply. Hence, there is an urgent need for the development of universal blood substitutes. One such blood substitute is glutaraldehyde-polymerized bovine hemoglobin. Despite the commercial development of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes, there has been little published work on the effect of reaction conditions on the physical properties of the polymerized hemoglobin dispersion. In this study, the effect of varying the concentration of the quenching agent NaBH4, glutaraldehyde concentration, and reaction time on the physical properties of PolyHb dispersions was studied by measuring the absolute molecular weight distribution, as well as oxygen-binding properties such as P-50, Hill coefficient, and methemoglobin level (MetHb) of these dispersions. Bovine hemoglobin was polymerized with glutaraldehyde using a parallel synthetic approach. Asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of the PolyHb dispersions. In general, high glutaraldehyde concentrations (20-30 times the molar concentration of hemoglobin) adversely affected the oxygen-binding properties of PolyHb dispersions, while NaBH4 concentrations (up to 300 times the molar concentration of hemoglobin) and reaction times (up to 24 h) did not appear to have any effect on the oxygen-binding properties of PolyHb dispersions.