Introduction: Stroke causes death and disability in patients throughout the world. At present, the only FDA- approved drug for ischemic stroke is recombinant tissue plasminogen activator (rt- PA). Unfortunately, rtPA can cause intracerebral hemorrhage and must use within limited time window (within ۳-۴.۵ hour after onset of stroke). Ultrasound with rtPA loaded liposomes (rtPA_L) was suggested as adjuvant therapy that reduce the dose of rt-PA and increase its efficiency. Different frequency protocols such as kHz and MHz were used for sonothrombolysis. In this study, we suggested a protocol including both kHz and MHz with rtPA_L The kHz wave could fragment rtPA_Ls and release rtPA, the MHz enhances lytic efficiency of released rt-PA by stable cavitation and its microstreaming. This protocol may reduce bioeffect of ultrasound wave and simultaneously at least maintain rt-PA lytic effect. Materials and Methods: The rt-PA loaded liposomes were prepared by hydrating lipid film with rt-PA and freezing liposomes under air pressure (۴ atm). Cylindrical human whole blood clots (۱ mm diameter) formed in and around micropipette at ۳۷ ˚C. In combination with rtPA_L ([rtPA]=۵۰ μg/ml) and human plasma, three protocol were used: ۱ MHz (۱.۵ W/cm۲_ ۳۰ minutes), ۱۳۰ kHz ( ۰.۰۱ W/cm۲_۱۰ s) + ۱ MHz (۰.۵ W/cm۲_ ۳۰ minutes) and control. Clots were imaged under microscope and clot lysis were quantified by measuring clot diameters before and after sonication. Results: Statistical analysis of clot diameter varation between two sonication groups (۱ MHz (۱.۵ W/cm۲_ ۳۰ minutes), ۱۳۰ kHz ( ۰.۰۱ W/cm۲_۱۰ s) + ۱ MHz (۰.۵ W/cm۲_ ۳۰ minutes)) do not show significant difference. Conclusion: Not significant difference between two sonication groups indicates that the dual frequency protocol by using less energy and consequently lower bioeffects may have similar effect as single frequency protocol.