The worldwide incidence of fatty liver disease continues to rise, which may account for concurrent increases in the frequencies of more aggressive liver ailments. Given the existence of histologically identical fatty liver disease subtypes, there is a critical need for the identification of methods that can classify disease and potentially predict progression. Herein, we show that a panel of protein kinase chemosensors can distinguish fatty liver disease subtypes. These direct activity measurements highlight distinct differences between histologically identical fatty liver diseases arising from diets rich in fat versus alcohol and identify a previously unreported decrease in p38α activity associated with a high-fat diet. In addition, we have profiled kinase activities in both benign (diet-induced) and progressive (STAM) disease models. These experiments provide temporal insights into kinase activity during disease development and progression. Altogether, this work provides the basis for the future development of clinical diagnostics and potential treatment strategies.