The staging of solid cancers is critical to the planning of both primary treatment with surgery and adjuvant therapies like chemotherapy; however, staging is not always possible with preoperative information and may require intraoperative evaluation of sentinel lymph nodes to confirm or disaffirm the presence of metastasis. Challenges are presented by standard-of-care sentinel lymph node dissection which must be quick and accurate enough to guide the surgical strategy despite a workflow that stretches from the operating room to the pathology lab; however, a solution is posed by fluorescence-assisted sentinel lymph node dissection which uses fluorescent probes to communicate the location and/or status of sentinel lymph nodes, reducing the complexity of the surgery and/or eliminating the need for rapid pathology. In support of this emerging modality, we have constructed a snapshot hyperspectral imaging system with sensitivity from the far-red to the near-infrared that enables sentinel lymph node dissection with multiple near-infrared fluorophores. We have also developed a spectral unmixing routine for in vivo quantification of the readily available fluorophores indocyanine green and methylene blue that can be extended to emerging fluorophores that actively target tumor cells. Both the imaging system and the unmixing routine have been tested in a clinical setting where they have successfully discriminated two dyes exhibiting different distributions.