Abstract: Provided is a method for simultaneously determining both [L]0 and Kd values of a ligand for a target protein. In one embodiment, the present technology involves performing quantitative equilibrium immunoassays at two different concentrations of the target and fitting the data to simultaneously determine Kd and [L]0. Also provided is a method for determining binding affinity of a pool of candidate ligands in a high-throughput manner. In another embodiment, the present technology method combines high-throughput nucleic acid sequencing with a display technology to obtain kinetic on-rates and off-rates, and thus Kd values, for the candidate ligands.