A PEGylated protein is prepared by conjugating polyethylene glycol (or PEG) with the protein, a process known as PEGylation. Most PEGylation processes lead to synthesis of different PEGylated forms of the protein, among which only one form is typically of interest. In this work, we propose a modeling and optimization-based approach for determining optimal operating conditions for protein PEGylation. To this end, a first-principles model is proposed and targeted experiments are carried out to estimate the model parameters. A simulation-based optimization is then carried out to suggest the best operating conditions. Specifically, results suggest that to maximize the concentration of mono-PEGylated product, the reaction should be carried out at high pH and with a high ratio of PEG to protein. Subsequent experiments are conducted to confirm the validity of the modeling and optimization approach.