Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX, and recombinant Factor IX during the course of a fed-batch bioreactor CHO cell culture. We describe Data Dependent Acquisition and Data Independent Acquisition (SWATH) Mass Spectrometry glycoproteomic workflows to efficiently capture details of the diverse post-translational modifications on Factor IX. We report two novel sites of glycosylation on human serum-derived Factor IX: an O-linked site at serine 141, and an N-linked site in the serine protease domain in a non-canonical consensus motif at asparagine 258. We observe diverse post-translational modifications on recombinant Factor IX, with a high level of heterogeneity that varies quantitatively over the time-course of fed-batch cell culture. Our detailed description of novel and diverse sites of glycosylation and other post-translational modifications provides a basis for future functional studies and contributes to structural characterisation of Factor IX in bioprocess control for the efficient production of effective therapeutic biosimilars and biobetters.