Tissue plasminogen activator (tPA) is the only approved drug for ischemic stroke in Canada but is limited in its clinical efficacy due to its short therapeutic window. This study sought to determine the effect of tPA on postnatal primary cortical neuron viability and aimed to identify the relevant cellular signalling mechanisms underlying the tPA-mediated effects in vitro. The data revealed that tPA significantly increased the propensity for cell survival within a time latency window of up to 3 hours. tPA-induced neuroprotective effects were significantly dependent upon the mTOR and JAK/STAT signalling pathways, while the MEK and PKA signalling pathways were found to play a less critical role. Immunocytochemical staining showed a marked increase in p-S6 expression following treatment with tPA, substantiating the vital role of mTOR activation in tPA-mediated neuroprotection. These results suggest the possibility of targeting the defined mechanisms to expand the therapeutic window of tPA in stroke recovery.