The development of biotherapeutic proteins requires the use of efficient analytical methods to support their manufacturing process and quality control (QC). Analytical approaches at intact and middle-up levels emerged as promising alternatives to bottom-up strategies for protein characterization as they require less sample amount and simplified sample handling, thus reducing the overall turn-around time. This study describes, for the first time, the development of an automated liquid chromatography-mass spectrometry (LC–MS) workflow comprised of an immobilized IdeS-HPLC column for on-line sample digestion, followed by a reversed-phase liquid chromatography (RPLC) for protein subunit separation, and a high-resolution MS for molecular weight analysis. A proof of concept study was described here for the characterization of a therapeutic mAb and a bsAb. For the mAb, this automated workflow enabled rapid characterization of post-translational modifications (PTMs) such as N-glycosylation, glycation and N-terminal lysine. For the bsAb, the same workflow was successfully employed to identify product impurities due to chain pairing. The sample analysis using this workflow can be accomplished within less than one day. This workflow demonstrated its potential as a multi-attribute method for characterization of therapeutic proteins.