Melanoma accounts for less than 5% of skin cancer cases, yet it causes more than 75% of skin cancer death, and its incidence is growing faster than any other cancer in the world. Because melanoma metastasizes early into regional/sentinel lymph nodes (SLN), SLN excision (SLNE) is probably the most important diagnostic procedure for melanoma patients, as histology provides the most relevant prognostic factor for the survival of melanoma patients. However, 50% of excised lymph nodes show no evidence of metastasis; and, current histological protocols involve sampling only a small portion of the SLN, resulting in a relatively high false negative rate. Furthermore, SLNE exposes the patients to complications such as swelling, edema and future risk of infection. Therefore, a clear need exists to improve the sensitivity and specificity of SLN analysis. Multispectral optoacoustic tomography (MSOT) ulitizes the molecular specificity of optical imaging, but capitalizes on the high temporal and spatial resolution of ultrasound imaging. This method allows sensitive detection of optical markers such as melanin, which would identify potentially metastatic lymph nodes, and indocyanine green (ICG), which can potentially label SLN. In this study, 148 lymph nodes were excised from 65 melanoma patients (stage I – IV), and the lymph nodes were examined by MSOT ex vivo to guide the pathologist to examination of melanin-containing regions of the lymph node, thereby doubling the detection rate. Compared to standard histology, MSOT demonstrated a superior 100% sensitivity/41% specificity. Further, 21 patients were scanned with a handheld MSOT device in vivo. MSOT was able to detect >90% of sentinel lymph nodes as compared to SPECT/CT, with the added ability of non-invasive assessment of the melanin status prior to excision. In vivo MSOT measurements were compared with ultrasound, SPECT/CT, planar fluorescence imaging and ex vivo histology, with a promising concordance between in vivo MSOT measurements and in vivo and ex vivo gold standard assessments. MSOT represents a viability modality to improve histological analysis of excised SLN in melanoma patients, and it offers the ability to stage lymph nodes noninvasively, potentially reducing the necessity to excise lymph nodes in some melanoma patients.