Heart failure (HF) poses a significant and enduring challenge in modern medicine, impacting over 6 million Americans and approximately 2% of the global adult population . Despite progress in pharmaceutical treatments and heart-supporting devices, HF patients continue to experience a high mortality rate due to the limited effectiveness of current pharmacological interventions . However, there is a potential breakthrough on the horizon within the field of cardiac gene therapy. This innovative approach involves utilizing adeno-associated virus (AAV) to overexpress specific cardiac genes in failing hearts for functional rescue. One of these potential gene therapy targets is cardiac bridging integrator 1 (cBIN1), a membrane scaffolding protein creating t-tubule membrane microdomains within cardiomyocytes to control beat-to-beat calcium transients and excitation-contraction coupling . In acquired human HF, t-tubule microdomains are disrupted due to reduced transcription of cBIN1, weakening calcium transients and impairing cardiac function. Fortunately, impaired microdomains can be restored via exogenous cBIN1 transduced by cardiac tropism AAV9, which has demonstrated effectiveness in reversing HF in animal models. We previously confirmed exogenous cBIN1 expression in mouse and pig cardiomyocytes following systemic delivery of AAV9-cBIN1. However, the transduction efficiency of AAV9 at a determined viral dosage (i.e. vg/kg) has a large batch-to-batch variability due to many confounding factors such as the presence of empty vectors, limiting our ability to compare dose-dependent therapeutic effects using different viral batches. To address this concern, here we developed a method to determine the in vitro transduction efficiency of AAV9-cBIN1 from various viral batches. We transduced HeLa cells with AAV9-cBIN1-V5, collected protein lysates after 24-hour transduction, and quantified cBIN1-V5 protein expression using Western blot analysis. Using this method, we were able to determine the effective AAV9 dosage from different batches. The ability to quantify cBIN1 transduction across different batches of AAV9 allows us to accurately titer the dose of AAV9-cBIN1 for its following usage in animal studies, ensuring the appropriate therapeutic quality.