Background: Patients with acute ischemic strokes frequently take an acetylsalicylic acid (ASA) premedication. We determined the impact of ASA on different thrombolysis strategies in vitro. Methods: For two clot types made from platelet-rich plasma (one with and one without ASA) lysis rates were measured by weight loss after 1 h for five different groups: in control group A clots were solely placed in plasma; in groups B and C clots were treated with rt-PA (60 kU/ml), and in groups D and E clots were treated with desmoteplase (DSPA; 2 μg/ml). Ultrasound (2 MHz, 0.179 W/cm2) was included in groups C and E. The fibrin mesh structures of the clots were investigated by electron microscopy. Results: For both clot types lysis rates increased significantly for all treatment strategies compared to their control group (each p < 0.001). The addition of ASA significantly increased the lysis rate in all 5 groups (each p < 0.001) and led to a ceiling effect concerning the treatment. A semiquantitative analysis of transmission electron micrographs revealed a decreased fibrin density for clots with ASA. For both clot types DSPA and ultrasound led to a significant dissolution of the fibrin mesh (both p = 0.029). Conclusions: In vitro ASA pretreatment leads to significantly increased lysis rates due to a weaker fibrin mesh in platelet-rich plasma clots.