Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. The covalent attachment of poly(ethylene glycol) molecules to proteins, PEGylation, has become one of the most successful technologies for the in vivo stabilization of protein drugs. In spite of many advances during the last three decades in the field of protein PEGylation, systematic studies addressing the effects of PEGylation on protein in vitro stability are lacking. These studies are necessary to understand the molecular mechanisms by which PEGylation stabilize proteins in vitro. The unifying theme of the work contained in this document can be described as the systematic study of the effects of PEGylation parameters, such as PEG size and PEGylation degree, on protein in vitro stability, and to offer mechanistic explanations for the observed effects. For these purpose several PEG-protein conjugates, with varying PEGylation degree and using PEG of different sizes, were synthesized. In Chapters 2 and 3, the effects of PEGylation on the enzymatic activity and in vitro stability of alpha-chymotrypsin (aCT), a model protein of the serine protease family, were studied. We found that PEGylation of aCT increased its kinetic stability, with slower inactivation rates when the enzyme was incubated at high temperatures. Also, PEGylation increased the thermodynamic stability of aCT, as evidenced by the higher T_m and free energies of protein unfolding of the PEG-aCT conjugates. Importantly, we were able to show, for the first time, that the thermodynamic stabilization of aCT inversely correlated with the protein structural dynamics measured by H/D exchange kinetics. In Chapter 4, the antimicrobial preservative-induced aggregation of the model protein alpha-chymotrypsinogen A (aCTgn) was prevented by PEGylation. The aggregation of aCTgn was only prevented when a high-molecular weight (5 kDa) PEG was used, whereas when low-molecular weight (700 Da) PEG was used, aCTgn still aggregated. This observation supports a mechanism in which the PEG acts as a molecular spacer sterically hindering protein-protein interactions that lead to aggregation.