Indocyanine green (ICG) is a clinically approved fluorescent dye that is used for lesion identification during hepatobiliary surgery. However, the chemical structure of ICG seems to be suboptimal for this excretion-based diagnostic readout. In this study the effect of systematic structural variations in Cy5-analogs were evaluated to elucidate the structure-activity relationship between hepatic uptake and excretion. Nine Cy5 dyes with variations in N-alkyl indole substitution (butyl sulfonate or methyl), and benzoannulation were synthesized in analogy to the ICG scaffold (Cy7.5-(SO3-)2). Photo-, bio- and chemical properties were analyzed and combined to identify structure-activity relationships using Spearman's correlation and multiparametric analysis. Chemical modifications were shown to alter (photo)chemical properties and constituted in clear biological effects; Sulfonate substitution supports hepatic excretion, while benzoannulation promoted nonspecific background accumulation. Cy5-(SO3-)2, rather than the ICG-analog Cy5.5-(SO3-)2, was identified as lead. Systematic evaluation revealed key structural determinants that influence biliary excretion and allowed lead selection of dyes for hepatobiliary imaging. These insights provide a foundation for the rational design of optimized fluorescent agents for this application.