The half-maximal effective concentrations (EC50) reported for specific antiviral agents often vary widely across in vitro studies. The cause for this may be due to differences in viral strains, cell types, and experimental design choices. Variability in EC50 estimates complicates potency interpretation and cross-study comparison. In this study, we applied a mechanism-based mathematical target cell-limited model to examine the impact of virus-specific parameters and different experimental design choices on EC50 estimation. Empirical EC50 values were estimated from simulated concentration–response experiments for different virus-specific properties and experimental conditions. Among viral parameters, EC50 changed more than 20-fold across the range of infection rate (β) and over 50-fold across viral production rate (ρ), whereas clearance rate had only minor effects. Among experimental factors, higher initial cell number (T₀) and later sampling time changed EC50 by more than 15-fold, while delayed time of adding drug increased EC50 by approximately 50 %. When both β and ρ were high, EC50 changed nearly 900-fold. Overall, our findings demonstrate that EC50 is highly sensitive to several virus-specific parameters and experimental design choices, and provides quantitative directions which may guide the design and interpretation of antiviral in vitro assays evaluating pharmacological activity.