PNGase F is an endoglycosidase that specifically removes N-linked glycans from glycoproteins. It is used extensively in workflows for characterizing N-linked glycan structures on therapeutic proteins and for identifying N-linked glycosylation sites in proteomic studies. We have expressed and purified recombinant PNGase F and shown that it is highly active against a variety of substrates, including purified glycoproteins and complex mixtures of human serum glycoproteins. Furthermore, the enzyme is compatible with the mass spectrometry workflow environment, including non-denaturing conditions and the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Finally, we show that recombinant PNGase F can be used to functionally deglycosylate therapeutic antibodies as measured by decreased antibody-dependent cell-mediated cytotoxicity after deglycosylation.