Monoclonal antibodies (mAbs) and related products undergo a wide range of modifications, many of which can often be directly associated to culture conditions during upstream processing. Ideally, such conditions should be monitored and fine-tuned based on real-time or close to real-time information obtained by the assessment of the product quality attribute (PQA) profile of the biopharmaceutical produced, which is the fundamental idea of process analytical technology. Therefore, methods that are simple, quick and robust, but sufficiently powerful, to allow for the generation of a comprehensive picture of the PQA profile of the protein of interest are required. A major obstacle for the analysis of proteins directly from cultures is the presence of impurities such as cell debris, host cell DNA, proteins and small-molecule compounds, which usually requires a series of capture and polishing steps using affinity and ion-exchange chromatography before characterization can be attempted. In the current study, we demonstrate direct coupling of protein A affinity chromatography with native mass spectrometry (ProA-MS) for development of a robust method that can be used to generate information on the PQA profile of mAbs and related products in as little as 5 min. The developed method was applied to several samples ranging in complexity and stability, such as simple and more complex monoclonal antibodies, as well as cysteine-conjugated antibody-drug conjugate mimics. Moreover, the method demonstrated suitability for the analysis of protein amounts of <1 μg, which suggests applicability during early-stage development activities.