Acute stroke, with ischemic stroke comprising 80% of all cerebrovascular incidents, has been recognized as one of the core problems in clinical medicine in need of prevention and treatment. Intravenous rtPA is the mainstay and the highest class evidence based method of acute ischemic stroke treatment, and is currently recommended 0-4.5 hours after stroke onset. In most patients decision on i.v. rtPA administration is striaghtforward, however the biggest concern is the symptomatic intracranial haemorrhage (sICH), which occurs in 3-7% of all treated patients, and is associated with worse 90-day functional outcome and higher disability than in those untreated. Finding a method of the powerful (highly specific and selective) identification of patients at highest risk of sICH, in order to increase the percentage of stroke patients safely treated with rtPA, is one of the most important challenges in stroke research. To address this problem we designed a major and complex project to identify blood, neuroimaging, and clinical biomarkers combined for prospective assessment of the risk of intracranial hemorrhage (ICH) after thrombolytic treatment of acute ischemic stroke. Herein, we reveal our general methodological approach with shortlisting of blood protein candidates selected with Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) that in the future might increase sensitivity and selectivity of the rtPA-associated sICH risk calculations.