We recently reported a new method for quantification of protein-ligand interaction by mass spectrometry, titration and H/D exchange (PLIMSTEX) for determining the binding stoichiometry and affinity of a wide range of protein-ligand interactions. Here we describe the method for analyzing the PLIMSTEX titration curves and evaluate the effect of various models on the precision and accuracy for determining binding constants using H/D exchange and a titration. The titration data were fitted using a 1:n protein:ligand sequential binding model, where n is the number of binding sites for the same ligand. An ordinary differential equation was used for the first time in calculating the free ligand concentration from the total ligand concentration. A nonlinear least squares regression method was applied to minimize the error between the calculated and the experimentally measured deuterium shift by varying the unknown parameters. A resampling method and second-order statistics were used to evaluate the uncertainties of the fitting parameters. The interaction of intestinal fatty-acid-binding protein (IFABP) with a fatty-acid carboxylate and that of calmodulin with Ca 2ϩ are used as two tests. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses novel aspects in modeling experimental titration data to determine the affinity of ligand binding. (PLIMSTEX). We demonstrated in that article that PLIMSTEX can be applied to determine the conformational change, binding stoichiometry, and affinity of a wide range of protein-ligand interactions including those that involve small molecules, metal ions, and peptides.Here we describe the method of modeling PLIMS-TEX titration curves and examine the effect of model modifications on the precision and accuracy that can be achieved when determining binding constants. A preliminary description of the modeling accompanied the first description of PLIMSTEX [9] as supplemental material, but since that time, we extended the model with more parameters, improved it by adding resampling, and tested it in a more complete manner. The interaction of intestinal fatty-acid-binding protein (IF-ABP) with a fatty-acid carboxylate and that of calmodulin with Ca 2ϩ are used as two test systems to demonstrate the H/D exchange titration method and the protein-ligand fractional species model. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses some novel aspects in modeling experimental titration data to determine the affinity of ligand binding.The basis for the modeling is nonlinear least squares (NLLS). Among all the curve fitting methods used in the biochemical literature, NLLS regression is probably the most common [10,11]. The "least squares" method was introduced by Legendre in 1805, but the first