Fluorescent proteins have found widespread applications for analysis of biological systems as they can be used to track various events within living cells. Multiple fluorescent proteins are also simultaneously used to monitor different aspects of biological systems. However, extensive overlap in the emission spectra of the fluorescent proteins poses challenges in extracting the contribution of individual proteins to overall fluorescence intensity measurements. This work addresses this issue by deriving a computational formulation for extracting the contribution of fluorescence intensities of individual reporters to the overall measurements taken using a plate reader. Then, this formulation is used for deriving an experimental design criterion for choosing sets of fluorescent proteins such that the accuracy of the estimated contribution of different fluorescent proteins is maximized. The results are validated using two sets of experimental data involving different sets of fluorescent proteins. This work represents the first quantitative study that evaluates experimental design for selection of fluorescent proteins to use simultaneously for multiple-labeling applications. (C) 2013 Elsevier Ltd. All tights reserved.