Human ST8 sialyltransferases are enzymes that transfer oligo- or polysialic acid to the non-reducing terminal glycans of proteins and lipids. This polysialic acid (PSA) is a chain of α2-8-linked sialic acid which has recently had increased attention from biomedical researchers because of PSA’s role in neurological diseases and tumour metastases. PSA is also extensively researched in biotechnology as a potential natural polymer to conjugate to therapeutic proteins to improve pharmacokinetics. To understand the mechanisms and constraints of polysialylation, we studied the six human ST8 sialyltransferases, and in particular ST8SIA2 and ST8SIA4, which are known to autopolysialylate. We expressed and purified these enzymes from HEK293 cell culture, and used mass spectrometry glycoproteomics to measure the site-specific glycosylation profiles and polysialylation sites of these autopolysialylated sialyltransferases.