SPR biosensing is a well-established tool for the investigation of binding kinetics between a soluble species and an immobilized (bio)molecule. While robust and accurate data analysis techniques are readily available for single species, methods to exploit data collected with a solution containing multiple interactants are scarce. In a previous study, our group proposed two data analysis algorithms for 1) the precise and reliable identification of the kinetic parameters of N interactants present at different ratios in N mixtures and 2) the estimation of the composition of a given mixture, assuming that the kinetic parameters and the total concentration of all interactants are known. Here, we extend the first algorithm by reducing the number of necessary mixtures. This is achieved by conducting experiments at different temperatures. Through the Van’t Hoff and Eyring equations, identifying the kinetic and thermodynamic parameters of N binders becomes possible with M mixtures with M comprised between 2 and N and at least N/M temperatures. The second algorithm is improved by adding the total analyte concentration as a supplementary variable to be identified in an optimization routine. We validated our analysis framework experimentally with a system consisting of mixtures of low molecular weight drugs, each competing to bind to an immobilized protein. We believe that the analysis of mixtures and composition estimation could pave the way for SPR biosensing to become a bioprocess monitoring tool, on top of expanding its already substantial role in drug discovery and development.