A method independent of the use of radioisotope monomers such as [(3)H]-thymidine was developed to evaluate the bioactivity of human epidermal growth factor (hEGF). Briefly, DNA synthesis of fibroblasts in hEGF-containing culture medium was evaluated via cell cycle experiments using flow cytometry; the DNA synthesis ratio was used to evaluate the bioactivity of hEGF. The unfolded fraction of hEGF, a physical parameter, was also evaluated by circular dichroism measurements. The hEGF with a higher unfolded fraction showed less bioactivity than hEGF from fibroblasts. There was a good relationship between the bioactivity of hEGF (i.e., DNA synthesis ratio) and the evaluated physical parameter of hEGF (i.e., unfolded fraction). The stability of hEGF solution containing several polymeric stabilizers during heat treatment was also determined by physical evaluation of the unfolded fraction and by bioactivity measurement of the DNA synthesis ratio. Heparin and a low molecular weight chemical of mannitol were effective stabilizers of hEGF during heat treatment.