mPEG-NHS is widely used in bioconjugation to modify proteins, peptides, and other biomolecules. It reacts efficiently with primary amines (–NH₂) present on lysine residues or N-termini of proteins, forming stable amide bonds. This modification increases the solubility and stability of the biomolecules, reduces immunogenicity, and extends their circulation half-life in therapeutic applications.

It is commonly employed in the development of PEGylated drugs, where attaching polyethylene glycol helps improve pharmacokinetics and reduce rapid clearance from the body. mPEG-NHS is also used in surface modification of nanoparticles, hydrogels, and medical devices to create non-fouling surfaces that resist protein adsorption and cell adhesion.

In research, it serves as a blocking agent in protein studies and helps in the preparation of modified enzymes and antibodies for diagnostic and imaging applications. Its ability to introduce a methoxy-terminated PEG chain makes it ideal for creating well-defined, mono-functional conjugates.