The advancement of biopharmaceutical manufacturing, particularly continuous processing, has heightened the need for next-generation analytical tools approaching real-time monitoring of critical quality attributes (CQAs) and process parameters (CPPs). Current methods, primarily offline and labor-intensive, fail at delivering analytical information that can be used for process analytical technology (PAT) to control and optimize the manufacturing process, while also lacking the ability of multi-attribute monitoring, thus requiring a large number of samples (or sampling amount) to be collected. This work introduces the concept of PAT-on-a-chip, consisting of an integrated microfluidic platform designed to perform at-line analysis and characterization of cell culture samples in the context of monoclonal antibody (mAb) production. Specifically, a sample preparation-free miniaturized lectin-based assay was developed to measure levels of high mannose glycans and integrated with affinity-based assays to measure mAb titers and key impurities, namely Chinese hamster ovary (CHO) host cell proteins (HCP), within the same chip, resorting to a common colorimetric readout. The microfluidic chips were operated in a customized and integrated instrument comprising miniaturized photodiodes, connected to a graphical user interface for data recording and signal quantification. The PAT-on-a-chip unit allowed to achieve fit-for-purpose analyte quantification, while offering performance comparable to state-of-the-art offline analytical methods (Pearson R > 0.93), namely capillary electrophoresis with laser-induced fluorescence (CE-LIF) for glycan analysis, well plate immunoassays for CHO HCP and protein A HPLC for mAb titers, thus validating its potential to expand the modern PAT toolbox.