Downstream purification of therapeutic antibodies requires candidates to be stable under various stress conditions, such as low pH. Current approaches to assess the conformational or colloidal stability of biologics may require significant amounts of material, and the testing may occur over an extended period of time. Furthermore, typical methodologies for early stability testing often do not directly address low pH stability, but focus more on conditions suitable for long-term drug product storage. Here we report a high-throughput method that measures protonation-induced unfolding of ligand binding sites for stability evaluation by surface plasmon resonance or PULSE SPR. This method, which requires only several micrograms of sample, is highly sensitive to the structural integrity of ligand binding sites and correlates well with thermal and chemical conformational stability. Combined with ligands targeting different regions of antibodies, this method allows a comprehensive assessment of antibody domain stability. By applying PULSE SPR, we found that antibodies with different complementarity-determining regions (CDRs), frameworks, formats, and interchain disulfide bonds showed different stabilities under acidic conditions. Additionally, biologics that aggregated in solution also had poor low pH stability. Taken together, PULSE SPR is a high-throughput and low sample consumption method that can be adopted to evaluate antibody domain stability and aggregation at low pH, which are two important aspects of therapeutic biologics.