Aspects of the present disclosure include methods for identifying a set of fluorophore-biomolecule reagent pairs for characterizing a sample by flow cytometry. Methods according to certain embodiments include calculating a spectral spillover spreading parameter for a plurality of fluorophores, pairing each fluorophore with a biomolecule that is specific for a biomarker of a cell in the sample to generate a plurality of fluorophore-biomolecule reagent pairs, generating an adjusted spillover spreading matrix for the fluorophore-biomolecule reagent pairs based on the spectral spillover spreading parameter of each fluorophore and a biomarker classification parameter and identifying an optimal set of fluorophore-biomolecule reagent pairs based on the calculated spillover spreading values from the adjusted spillover spreading matrix. Systems and non-transitory computer readable storage medium for practicing the subject methods are also provided.