Tissue plasminogen activator (tPA) is the only FDA-approved medical therapy for acute ischemic stroke. But as a serine peptidase, intravenous tPA can affect the expression of other proteases that may be implicated in blood-brain barrier breakdown. Such parallel cascades of cell signaling may be involved in intracranial hemorrhage, the major side effect of tPA. Here, we describe an initial attempt in proteomic substrate profiling, i.e., degradomics in human plasma within the context of acute stroke. Plasma from acute stroke patients were analyzed pre- and post-intravenous tPA using tandem mass spectrometry and protein array profiling to identify substrates and proteases of interest. In non-tPA-treated stroke plasma, degradomic patterns indicated a rapid induction of protease activity within 3 h of stroke onset that mostly stabilized by 24 h. But in tPA-treated patients, pre- and post-tPA samples from the same patient demonstrated distinct degradomic patterns that persisted even up to 3-5 days after stroke onset. Matching control patients without strokes had little change in degradomic profiles over time. Our findings demonstrate that tPA treatment changes the plasma degradomic profiles in acute stroke patients. These composite proteolytic profiles may provide a glimpse of the pleiotropic effects of tPA on cellular signaling cascades at the bedside. This study supports the feasibility of performing pharmaco-proteomics at the bedside, which may ultimately allow us to dissect mechanisms of thrombolysis-related therapeutic efficacy in stroke.