Computational methods for the estimation of stoichiometric association constants for multiple-ligand binding systems are currently based on non-linear least-squares regression analysis. These computational methods require sophisticated, iterative algorithms to assure convergence to a solution, as well as initial parameter and error estimates. A simple procedure, called lambda-invariance testing (LIT), was developed that provides a single-pass (non-iterative) estimation of stoichiometric association constants. The LIT method was applied to simulated binding data containing Gaussian error and to real data drawn from the literature. This method provided parameter estimates essentially equivalent to those obtained by least-squares regression analysis, with no initial parameter or error estimates required.