PEGylation, the covalent attachment of poly(ethylene glycol) (PEG) to biomolecules, is a well-established molecular modification strategy that enhances the pharmacological properties of conjugated proteins by improving stability, reducing immunogenicity, extending half-life, and increasing solubility. Despite its benefits, PEGylated proteins are often heterogeneous mixtures containing positional isoforms that are challenging to eliminate and can significantly impact the biological activity of drugs. This study developed a reversed-phase ultra-performance liquid chromatography (RP-UPLC) method for the separation and quantification of positional isoforms in commercial PEGylated recombinant human growth hormone (PEG-rhGH) at the peptide level. In this strategy, PEG-rhGH was digested into peptides via trypsin treatment and then separated and quantified using RP-UPLC. Under the chromatography condition optimized from Quality by Design (QbD) principles, this method can effectively distinguish the PEGylated peptides in five isomers. MALDI-TOF MS, SDS-PAGE and Edman degradation indicated that each of these five isomers was conjugated with one 40 kDa branched PEG at a specific lysine residue and the N-terminal. Through the concentration correction of PEGylated peptides varying in sequence lengths, the proportion of five modified sites can be quantified. The comparative assay showed that this strategy effectively addresses the limitations of the commonly used IEC method, especially under challenging conditions like post-translational modifications (e.g., oxidation, deamidation, and cyclization) and conformational changes. As a result, it offers improved robustness and accuracy. Furthermore, the proposed RP-UPLC method exhibited outstanding repeatability across various storage conditions, making it a reliable tool for quality control and product release in the biopharmaceutical industry.