The increasing demand on biopharmaceuticals is pressuring the industry to produce more and better biotherapeutics. Product development optimization approaches that measure product quality and quantity after purification do not consider the pool of defective biosynthesized product that fails to purify. Thus, these approaches underestimate productivity and cannot be used to identify bottlenecks in production. To resolve this problem, we developed a suite of proteomic techniques that assess both quality (post-translational modifications, PTMs) and relative quantity of biotherapeutic proteins during culture. We optimized these workflows to eliminate polymers such as Pluronic F68, commonly added to mammalian cell suspension cultures. These workflows combine a detailed characterization of PTMs in the purified protein (using Byonic (Protein Metrics)) with SWATH-MS measurement of the protein relative abundance and its PTMs during bioreactor production. We focused on a difficult-to-produce biotherapeutic: Coagulation Factor IX (FIX). FIX is N-glycosylated, O-glycosylated, Beta-hydroxylated, phosphorylated, sulfated, disulphide-bonded, and gamma-carboxylated (FIX’s most important PTM, and the most difficult to detect by MS). Here, we compared FIX produced during Fed-batch (using two different media supplements) and perfusion bioreactors (rFIX). We analysed the relative quantities of rFIX and its PTMs in daily supernatant samples and in purified rFIX. We: 1) measured relative abundance of rFIX and host cell proteins by SWATH-MS in bioreactor supernatants and in purified material; 2) identified new glycosylation, sulfation/phosphorylation, and oxidation sites in FIX (interestingly, most of the new PTMs in rFIX were at/close to the gamma carboxylated domain, which is critical for FIX function); 3) generated a PTM-focused ion library using Byonic’s information to measure relative abundance of rFIX PTMs during bioreactor operation and in purified FIX; and 4) performed relative quantification of derivatized gamma carboxylation by SWATH-MS. These workflows can be adapted to any protein of interest, produced with any biological platform, during any fermentation conditions.