Novel human interferon alpha 2b (hIFN<i>α</i>2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested <i>in vivo</i> for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFN<i>α</i>2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFN<i>α</i>2b ORF were overexpressed in <i>Escherichia coli</i> BL21 (DE3) resulted in three muteins: hIFN<i>α</i>2b C2D, hIFN<i>α</i>2b C99D, hIFN<i>α</i>2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc^99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFN<i>α</i>2b C2D C99D > hIFN<i>α</i>2bC2D > hIFN<i>α</i>2bC99D > wild type. hIFN<i>α</i>2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFN<i>α</i>2b seems to be responsible for the fate of hIFN<i>α</i>2b <i>in vivo</i>