<b><i>Introduction:</i></b> Tranexamic acid (TXA) is the standard medication to prevent or treat hyperfibrinolysis. However, prolonged inhibition of lysis (so-called “fibrinolytic shutdown”) correlates with increased mortality. A new viscoelastometric test enables bedside quantification of the antifibrinolytic activity of TXA using tissue plasminogen activator (TPA). <b><i>Materials and Methods:</i></b> Twenty-five cardiac surgery patients were included in this prospective observational study. In vivo, the viscoelastometric TPA test was used to determine lysis time (LT) and maximum lysis (ML) over 96 h after TXA bolus. Additionally, plasma concentrations of TXA and plasminogen activator inhibitor 1 (PAI-1) were measured. Moreover, dose effect curves from the blood of healthy volunteers were performed in vitro. Data are presented as median (25–75th percentile). <b><i>Results:</i></b> In vivo TXA plasma concentration correlated with LT (<i>r</i> = 0.55; <i>p</i> &#x3c; 0.0001) and ML (<i>r</i> = 0.62; <i>p</i> &#x3c; 0.0001) at all time points. Lysis was inhibited up to 96 h (LT_TPA-test: baseline: 398 s [229–421 s] vs. at 96 h: 886 s [626–2,175 s]; <i>p</i> = 0.0013). After 24 h, some patients (<i>n</i> = 8) had normalized lysis, but others (<i>n</i> = 17) had strong lysis inhibition (ML &#x3c;30%; <i>p</i> &#x3c; 0.001). The high- and low-lysis groups differed regarding kidney function (cystatin C: 1.64 [1.42–2.02] vs. 1.28 [1.01–1.52] mg/L; <i>p</i> = 0.002) in a post hoc analysis. Of note, TXA plasma concentration after 24 h was significantly higher in patients with impaired renal function (9.70 [2.89–13.45] vs.1.41 [1.30–2.34] µg/mL; <i>p</i> &#x3c; 0.0001). In vitro, TXA concentrations of 10 µg/mL effectively inhibited fibrinolysis in all blood samples. <b><i>Conclusions:</i></b> Determination of antifibrinolytic activity using the TPA test is feasible, and individual fibrinolytic capacity, e.g., in critically ill patients, can potentially be measured. This is of interest since TXA-induced lysis inhibition varies depending on kidney function.