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Introduction: While methods for decreasing door-to-needle (DTN) time with thrombolytics for acute ischemic stroke (AIS) have been explored, there has not been investigation into strategies to mitigate medication waste. Increased preference of tenecteplase (TNK) over alteplase has resulted in concerns over excess medication waste costs, as prior to FDA approval of TNK for AIS in March 2025, no reimbursement program was available for this medication. Objectives: This study aimed to characterize rates of TNK waste across four urban hospitals including a Comprehensive Stroke Center (CSC), Thrombectomy Certified Center (TSC), and two Primary Certified Centers (PSCs) when utilized for AIS in the emergency department (ED). Secondary objectives included identification of risk factors associated with increased TNK waste and associated US dollar cost-burden. Methods: This multi-center, retrospective cohort study included patients aged ≥18 years with AIS symptoms seen in the ED at one of four certified stroke centers for whom TNK was ordered between August 2021 and September 2024. A logistic regression model was used to assess risk factors associated with TNK waste. The value of wasted doses was calculated for each site based on median time from order to delivery of the medication, pharmacy compounding model and associated labor costs, and average US wholesale product price of $9,900 per 50 mg vial provided by Lexidrug. Results: Of 714 patient encounters, there were 55 (7.9%) total wasted doses across four hospitals. After adjusting for stroke vs no stroke diagnosis, NIHSS at admission, and last known well (LKW) to arrival, pharmacist bedside mixing (OR = 0.11 [0.02, 0.37]; p = 0.003) and higher NIHSS scores at admission (OR = 0.94 [0.90, 0.99]; p = 0.02) were associated with significantly lower odds of TNK waste. Confirmed AIS or transient ischemic attack (TIA) vs no stroke diagnosis (OR = 9.02 [4.43, 18.20]; p <0.001) and longer LKW to arrival times (OR = 1.05 [1.02, 1.10]; p = 0.01) were associated with significantly higher odds of TNK waste. The most common reasons for waste were rapid or early improvement, care team unable to determine eligibility, recent surgery, or prior stroke in the past 3 months. The estimated cost of TNK waste over the study period was $544,729.35. Conclusion: Pharmacist bedside mixing of TNK may lead to lower rates of TNK waste, reducing overall cost burden to the healthcare system.