Near-infrared fluorescence (NIRF) imaging involves the separation of weak fluorescence signals from backscattered excitation light. The measurement sensitivity of current NIRF imaging systems is limited by the excitation light leakage through rejection filters. In this contribution, the authors demonstrate that the excitation light leakage can be suppressed upon using appropriate filter combination and permutations. The excitation light leakage and measurement sensitivity were assessed and compared in this study by computing the transmission ratios of excitation to emission light collected and the signal-to-noise ratios in well-controlled phantom studies with different filter combinations and permutations. Using appropriate filter combinations and permutations, we observe as much as two orders of magnitude reduction in the transmission ratio and higher signal-to-noise ratio.