The first detailed examination of flow injection-capillary electrophoresis (FI-CE) active parameters and their interactions via response surface methodology (RSM) is presented. Specifically, RSM in the form of a Box-Behnken design was implemented to effectively predict the significance of capillary length, voltage and injection volume on the optimization of an in-house built FI-CE analyzer. Initial studies were performed assessing peak height and peak shape of the model compound N,N-dimethylformamide. Optimum model conditions were then derived and used in the model separation of two small molecules, nicotinamide adenine dinucleotide, reduced form (NADH) and benzenesulfonamide. By implementing the RSM approach, detailed examination of active FI-CE parameters was possible, including the ability to reveal a significant interactive effect. This work is not only highly significant for advancing FI-CE developments, but instructive for investigators actively exploring other coupled analytical techniques and associated experimental parameters.