Poly(ethylene glycol) (PEG) has been widely used to prolong the residence time of proteins in blood and to decrease their immunogenicity and antigenicity. A drawback of this polymer lies in its polydispersity that makes difficult the identification of the sites of protein modification. This is a mandatory requirement if a PEGylated protein should be approved as a drug. Here, a fast and reliable method is proposed to characterize proteins conjugated at the level of glutamine (Gln) residues using microbial transglutaminase (TGase). The novelty resides in the use of a monodisperse Boc-PEG-NH(2) for the derivatization that allows the direct identification of the sites of PEGylation by electrospray ionization mass spectrometry (ESI-MS). The procedure has been tested on three model proteins, namely, human granulocyte colony-stimulating factor, human growth hormone, and horse heart apomyoglobin. The Gln residues linked to the polymer chain were easily identified by ESI-MS and tandem MS analyses, demonstrating the advantage of using a monodisperse polymer in combination with mass spectrometry for an easy characterization of conjugated proteins. Interestingly, the PEGylation reaction led to the production only of mono- and bis-derivative products, indicating that the TGase-mediated PEGylation can be extremely selective and thus very useful for the derivatization of protein drugs.