OBJECTIVE Near-infrared fluorescence (NIRF) imaging using exogenous contrast has gained much attention as a technique for enhancing visualization of vasculature using untargeted agents, as well as for the detection and localization of cancer with targeted agents. In order to address the emerging need for standardization of NIRF imaging technologies, it is necessary to identify the best practices suitable for objective, quantitative testing of key image quality characteristics. Toward the development of a battery of test methods that are rigorous yet applicable to a wide variety of devices, we have evaluated techniques for phantom design, measurement, and calculation of specific performance metrics. METHODS Using a NIRF imaging system for indocyanine green imaging, providing excitation at 780 nm and detection above 830 nm, we explored methods to evaluate uniformity, field of view, spectral crosstalk, spatial resolution, depth of field, sensitivity, linearity, and penetration depth. These measurements were performed using fluorophore-doped multiwell plate and high turbidity planar phantoms, as well as a 3D-printed multichannel phantom and a USAF 1951 resolution target. CONCLUSIONS Based on a wide range of approaches described in medical and fluorescence imaging literature, we have developed and demonstrated a cohesive battery of test methods for evaluation of fluorescence image quality in wide-field imagers. We also propose a number of key metrics that can facilitate direct, quantitative comparison of device performance. These methods have the potential to facilitate more uniform evaluation and inter-comparison of clinical and preclinical imaging systems than is typically achieved, with the long-term goal of establishing international standards for fluorescence image quality assessment.