The covalent attachment of poly(ethylene glycol) (PEG) to therapeutically active proteins (PEGylation) has become an important method to deal with the pharmacological difficulties of these polypeptides, such as short body-residence times and immunogenicity. However, the derivatives of PEG used for PEGylation lack further functional groups that would allow the addition of targeting or labeling moieties. Squaric acid diethyl ester was used for the chemoselective single-step activation of poly(ethylene glycol)s into the respective ester amides. The resultant selective protein-reactive poly(ethylene glycol)s were investigated with respect to their selectivity towards amino acid residues in bovine serum albumin (as a model protein). The presented procedure relies on a robust two-step protocol and was found to be selective towards lysine residues; the activated polyethers are efficient and stoichiometric PEGylation agents with a remarkable hydrolytic stability over a period of several days. By adjusting the pD value of the conjugation mixture, the chemoselectivity of the activated PEGs towards the a- and e-amino groups of lysine methyl ester was effectively changed.