Background: A subset of benign Spitz nevi with atypical features is currently nearly impossible to differentiate from potentially fatal melanoma. Spitz nevi predominantly occur on the head or extremities of children representing a reconstructive challenge. Over treatment of Spitz nevi as melanoma results in significant morbidity while under treatment of melanoma is fatal. Objective: Previous data has shown that NEDD9 and HMB45 proteins are differentially expressed in nevi and melanoma. We hypothesized that the subcellular localization of HMB45 and NEDD9 may be helpful to classify benign nevi and melanoma. Furthermore differential localization of these biomarkers were applied to Spitz tumors to address this diagnostic dilemma. Methods: Automated Quantitative Analysis (AQUA) was used to quantify protein expression levels in subcellular compartments using fluorescence-based immunohistochemistry. The subcellular AQUA ratio for a sample was defined as the natural log of the AQUA score in the nuclear compartment divided by the non-nuclear compartment. Tissue Microarrays (TMA) evaluated nevi and malignant melanoma. Whole section analysis evaluated the diagnostic utility of the subcellular AQUA ratio in Spitz tumors. Results: NEDD9 subcellular AQUA ratio was significantly reduced in primary melanoma (n=19) versus nevi (n=17) on our TMA cohort (p=0.0086), significantly reduced in melanoma metastases (n=37) versus nevi (n=220) on the Yale Nevi TMA (p < 0.0001), and significantly reduced in primary melanoma (n=112) and melanoma metastases (n=128) versus nevi (n=54) in SPORE84 melanoma progression array (p=0.0003, Tukey post-hoc < 0.05). HMB45 subcellular AQUA ratio was significantly reduced in primary melanoma (n=19) versus nevi (n = 19) on Yale TMA (p=0.0001). On whole section analysis, the HMB45 and NEDD9 subcellular AQUA ratio shared a similar distribution between Spitz nevi (n=20), atypical Spitz nevi (n=20) and Spitzoid melanoma (n=9). Conclusions: Subcellular localization using the subcellular AQUA ratio of HMB45 and NEDD9 shows promise for distinction of nevi from melanoma on TMA but is not useful in discriminating between Spitz nevi and melanoma with Spitzoid features. The maximum HMB45 AQUA score of a whole tissue section was promising on discovery analysis at differentiating between Spitz nevi and melanoma with Spitzoid features (p=0.007, ROC area 0.711) but requires validation on an independent cohort.