To apply a systems pathology-based approach to the quantification of nuclear Active β-catenin and human leukocyte antigen class I, and assess the biomarker involvement in a cohort of prostate tumor patients. The systems pathology approach applied allows a precise quantification of the marker expression in the different cell compartments as well as the determination of the areas that coexpress two markers. Our data shows that the accumulation of β-catenin in the nuclear compartment is significantly decreased in the adjacent normal areas when compared to tumor of the same patients (p < 0.001). In conclusion, the application of this novel multiple immunofluorescence assay demonstrates that the upregulation of Active β-catenin is a relatively common feature of prostate tumor development, and further supports the activation of the Wnt/β-catenin pathway in prostate cancer progression. The online version of this article (10.1186/s13104-019-4100-z) contains supplementary material, which is available to authorized users.