Purpose. We successfully developed recombinant human interferon alpha-2b (rhIFN-α2b) and mutein forms through the site-directed mutagenesis technique. The mutein forms were developed by substituting cysteins at positions 2 and 99 with aspartic acids. The potential adverse effects of these rhIFN-α2bs were assessed by acute and subchronic studies. Methods. In the acute study, rhIFN-α2bs were subcutaneously administered to mice at a single dose of 97.5 μg/kg, 975 μg/kg, and 9.75 mg/kg BW and were observed for 14 days. In the subchronic study, single dose of 1.95 μg/kg and 19.5 μg/kg, respectively, was given subcutaneously every 3 days for 45 days. Results. No death as well as abnormality in body weight, behavior, presentation of main organs, and value of plasma SGPT and SGOT was observed. Wild type and mutein rhIFN-α2bs did not show significant adverse effects at dose up to 9.75 mg/kg BW. Administration of these rhIFN-α2bs given repeatedly did not induce any adverse effect. Conclusion. These results suggest that our rhIFN-α2bs are safe. However, further study is still needed to clarify the safety issue before use in clinical trial.