Biopharmaceuticals market, including monoclonal antibodies (mAbs), therapeutic proteins and vaccines has been expanding recently and expect to continue to grow. In comparison to synthetic drugs, which most of them have low molecular weight, biopharmaceuticals have high molecular mass and complex structure in general. More over, heterogeneity could be generated by post-translational modification in upstream and artificial modification in downstream processing. Characterization of biomolecules including heterogeneity is quite important in R&D and QC in biopharmaceuticals and methods by HPLC is typically used. In this presentation, I will present new applications to heterogeneity analysis of mAb and PEG protein by HPLC including analysis of vaccine using several separation modes like Size-exclusion chromatography, Ion-exchange chromatography, Hydrophobic interaction chromatography. Also I will introduce brand-new size-exclusion chromatography columns for mAb separation and reversed-phase chromatography column for protein separation.