Lysosomes contain multiple proteases, which play a crucial role in breast cancer invasion and metastasis. Noninvasive labeling of lysosomes in breast cancer cells and solid breast tumor models is therefore useful to study lysosomal trafficking and its role in invasion. We have synthesized a novel compound, 6'-O-lissamine-rhodamine B-glucosamine, to fluorescently label lysosomes, and evaluated the compound in human breast cancer cells in cell culture or in orthotopic human breast cancer models. We demonstrated that this novel compound biosynthetically labeled lysosomal proteins following addition to cell culture medium or following intravenous injection into mouse models of breast cancer. Fluorescence from 6'-O-lissamine-rhodamine B-glucosamine colocalized with several well-established lysosomal markers, such as lysosome-associated proteins 1 and 2 (LAMP-1 and -2) and CD63. We also demonstrated the feasibility of performing in vivo fluorescence imaging of 6'-O-lissamine-rhodamine B-glucosamine to image lysosomes in human breast cancer models.