Temporal lobe epilepsy (TLE) is one of the most common forms of drug-resistant epilepsy and its precise etiology is still unknown making it imperative to understand the molecular pathways contributing to the disease. It is, therefore, crucial to study the therapeutic targets and signalling pathways involved in the treatment of this disease. Pyk2 has previously been shown to be activated in stress-mediated pathways in the brain. Consequently, we hypothesized that Pyk2 activation might contribute to the pathogenesis of the disease. A pilocarpine model of TLE was used to examine p-Pyk2Tyr402 protein levels as well as mRNA levels of Pyk2 in the cortex, hippocampus, and anterior temporal lobe. Immunofluorescence analysis confirmed its presence in certain cell types. Further, we performed a calcium assay using flow cytometry in order to determine if calcium is involved in its activation. A significant increase in pPyk2Tyr402 was found in the hippocampus and ATL regions of the TLE model which was positively correlated with calcium levels. Additional evidence of Pyk2 activation was obtained in epileptic patient samples. Pyk2 was found to be activated region-specifically in TLE for the first time, emphasizing the role it plays in epileptogenesis and the generation of independent epileptogenic networks. Further, the overactivation of Pyk2 suggests that it can be a potential therapeutic target with anti-epileptogenic effects.