Sialic acid expressed as an outer terminal units on a therapeutic glycoprotein play immunological and physiological roles such as immunological processes, hormonal response, and signal transmission. In particular, sialic acid as a N-Glycolylneuraminic acid (Neu5Gc), which is not synthesized in human due to an inactivated CMAH gene, often observed in therapeutic glycoproteins produced from mammalian cell lines. Exogenous Neu5Gc can be metabolically incorporated into human cells and the Neu5Gc antibodies which lead immune response will be produced and circulated through blood. Therefore, the determination for a non-human sialic acid of biotherapeutics produced in mammalian cell is highly required for drug’s safety i.e. immunogenicity. In this study, we developed an analytical tool using mass spectrometry to selectively identify and quantify Neu5Gc in biotherapeutics including Erythropoietin, Inflixmab, and Interferon-β with high sensitivity. Each therapeutic glycoprotein was enzymatically treated with PNGase F to release N-glycans, followed by chemical hydrolysis to liberate sialic acids (Neu5Ac and Neu5Gc). The Neu5Gc was chromatographically separated from Neu5Ac on PGC column. MRM transitions and instrument parameters of UHPLC/triple quadrupole (QqQ) MS were optimized with Neu5Gc standard. The limits of detection/ quantitation (LOD/ LOQ) for Neu5Gc and the linearity between Neu5Gc concentration and MS signal for quantitation were also examined. The concentration of a non-human glycan (Neu5Gc) from various therapeutic glycoprotein content was determined at low nano mole levels. This method will be a valuable platform for not only QbD processes of biotherapeutics but also QA/QC of products.