BackgroundUse of molecular‐based diagnostics for companion animals is impeded by availability of technology platforms, tissue acquisition requirements, and species‐specific reagents.Hypothesis/ObjectivesTo validate a quantitative nuclease protection assay (qNPA) to simultaneously measure RNA expression of multiple genes in archived formalin‐fixed paraffin‐embedded (FFPE) tumors from dogs.AnimalsAll tumor biopsy samples were collected retrospectively from surgical biopsies and in the care of veterinarians.MethodsRetrospective case series. A qNPA 96‐well ArrayPlate was built using 30 canine‐specific genes, 5 housekeeping genes, positive and negative controls with qualified gene‐specific oligonucleotides. Pearson's correlation, coefficient of variation (CV), and multivariate analysis were used to determine analytical performance using 40 FFPE dog tumors. Once validated, 70 FFPE dog tumors were analyzed for differences in gene expression using hierarchical clustering and analysis of variance of log transformed data. Immunohistochemistry (IHC) was performed to correlate gene expression and protein expression in a subset of tumors.ResultsThe assay was linear with decreasing sample input (R 2 = 0.978), reproducible within and between 96‐well plates (r = 0.988 and 0.95, respectively) and between different laboratories (CV = 0.96). Hierarchical cluster analysis showed grouping of tumors by histogenesis and oncogenes. Significant differences were found between BCl2, E2F transcription factor 1, MDM2, COX‐2, MET proto‐oncogene receptor kinase, and other biologically relevant gene expression in tumor subtypes. Immunohistochemistry confirmed protein expression.Conclusions and Clinical ImplicationsBecause this technology works reliably on FFPE specimens, it can help expedite the broad introduction of multiplexed genomic information for improved diagnostics and discovery of new targets for therapies in veterinary oncology.