Novel protein therapeutics have become increasingly important modalities for treating diseases. Such therapeutics include recombinant fusions of pharmacoactive polypeptides to half-life extenders such as monoclonal antibodies, fragments of antibodies, and albumin. Half-life extension can also be achieved via chemical attachment to polymers such as polyethylene glycol. Any of these therapeutics may be susceptible to biotransformation, most notably in vivo proteolytic truncation, and it is vital to understand this phenomenon during early drug development to ensure correct pharmacokinetic profiling and optimize the in vivo stability through re-engineering. In this paper, we describe an integrated approach that combines differential enzyme-linked immunosorbent assay (ELISA) with ligand-binding-mass spectrometry (LB-MS) to provide a thorough understanding of the biotransformation of novel protein therapeutics. Differential ELISA allows for a fast, high-throughput means to reveal gross in vivo proteolytic liabilities. Ensuing LB-MS analysis provides higher resolution details such as specific vulnerable loci to allow design refinement of the molecule. In this work, the power of the approach is elucidated by application to the optimization of a promising drug candidate, FGF21.