This article focuses on the methods for determining vaccine potency, especially the feasibility and challenges of replacing traditional in vivo methods with in vitro methods, as well as related considerations. The in vivo method for determining vaccine potency has been used for a long time for vaccine quality control, but it has problems such as high variability, low experimental efficiency, and animal ethics issues. With the 3R principle and technological progress, in vitro methods have gradually become a trend to replace in vivo methods. In vitro testing methods have advantages in evaluating the consistency of vaccine production and monitoring changes during the production process, and can more effectively evaluate the stability and immune effects of vaccines. Although the mechanisms of in vitro and in vivo methods are different, and the quality control attributes of the two methods are not completely consistent, in vitro methods can still effectively replace in vivo potency assays, especially for quality control during the early stages of vaccine development. Common in vitro alternative methods include immunoassay, physicochemical analysis, and molecular biological assay. The selection of appropriate methods depends on the type of vaccine and its mechanism of action. In addition, the article emphasizes that the establishment of in vitro methods should be included in the vaccine development process as early as possible, and ensure its stability, reproducibility, and correlation with clinical efficacy. Although the promotion of in vitro alternative methods faces challenges, with technological progress, it is expected to further replace animal experiments in the future, improving the efficiency and ethicality of vaccine quality control.