The determination of equilibrium dissociation constant (K_d) alongside the maximum binding capacity (Bmax) of protein-ligand interactions is essential in understanding binding affinities and binding capacities, which plays a major role in the design and development of new therapeutic molecules. Rapid and accurate determination of these biophysical parameters (k_d and Bmax) in noncovalent protein – ligand interactions is desirable in the modern drug discovery process. Previously, we demonstrated detection of noncovalent protein-ligand complexes by infrared-assisted matrix desorption electrospray ionization mass spectrometry (IR-MALDESI-MS). Here, we report the first determination of biophysical parameters from noncovalent protein - ligand interactions using a ligand titration approach by IR-MALDESI-MS. Unlike conventional electrospray ionization-mass spectrometry (ESI-MS), IR-MALDESI-MS enables analysis in < 13 s per ligand concentration, emphasizing its speed, automation, and sensitivity. Native mass spectrometry by IR-MALDESI was performed on carbonic anhydrase II (CAH) incubated with sulfanilamide (SLFA), which is a known inhibitor. Coupled with other published studies, these results demonstrate that IR-MALDESI has strong potential as a high-throughput screening (HTS) technique for rapidly and accurately determining the K_d and Bmax of protein-ligand complexes.