Handheld-probe-based optical imagers are a popular approach toward breast imaging because of their potential portability and maximum patient comfort. A novel handheld-probe-based optical imager has been developed and its feasibility for three-dimensional fluorescence tomographic imaging demonstrated. Extensive tomography studies were performed on large slab phantoms (650 ml) to assess the performance limits of the handheld imager. Experiments were performed by using different target volumes (0.1-0.45 cm3), target depths (1-3 cm), and fluorescence (Indocyanine Green) absorption contrast ratios in a nonfluorescing (1:0) and constant fluorescing backgrounds (1000:1 to 5:1). The estimated sensitivity and specificity of the handheld imager are 43% and 95%, respectively.