Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences which could exist between human patients and animal models. Little is currently known about how kinase expression can vary between species, and tools to quantitatively measure such differences are lacking. We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog and cynomolgus monkey). These methods were then used to profile inter-species kinome variability in spleen with three of the current techniques used in targeted proteomics (MRM, PRM and IS-PRM). IS-PRM provided the highest number of kinase identifications, and the results indicate that while this initial set of kinases exhibits high correlation between species for this tissue type, discreet species-specific differences do exist, especially within the cyclin-dependent kinase (CDK) family. Understanding these differences could help rationalize the findings of preclinical studies and have major implications for the selection of these animals as models in kinase drug development.