CAP256V2LS, a broadly neutralizing monoclonal antibody (bNAb), is being pursued as a promising drug for HIV-1 prevention. The total level of tyrosine-O-sulfation, a post-translational modification, was known to play a key role for antibody biological activity. More importantly, here wedescribe for the first time the significance of the tyrosine-O-sulfation proteoforms. We developed a hydrophobic interaction chromatography (HIC) method to separate and quantify different sulfation proteoforms, which led to the direct functionality assessment of tyrosine-sulfated species. The fully sulfated (4-SO) proteoform demonstrated the highest in vitro relative antigen binding potency and neutralization efficiency against a panel of HIV-1 viruses. Interestingly, highly variable levels of 4-SO were produced by different clonal CHO cell lines, which helped the bNAb process development towards production of a highly potent CAP256V2LS clinical product with high 4-SO proteoform. This study presents powerful insight for any biotherapeutic protein development where sulfation may play an important role in product efficacy.