Purpose: To investigate the test-retest reliability and concurrent validity of 2MST for assessing the physical performance of patients with total knee arthroplasty (TKA). Materials and methods: Fifty-one patients with TKA, aged more than 18 years, of both sexes, and underwent a primary TKA at least six months ago, were included in this study. The intraclass correlation coefficient (ICC) and 95% confidence interval (CI), standard error of measurement (SEM), and minimum detectable change at the 95% confidence level (MDC95) were used to determine the reliability of 2MST. In the validity, the correlations of the 2MST between the six-minute walk test (6MWT) and the two-minute walk test (2MWT) were assessed. Results: The reliability of 2MST was excellent (ICC = 0.97, SEM = 2.76, MDC95 = 7.6). There was a moderate correlation between 2MST and 6MWT (r = 0.586; p = 0.000), as well as between 2MST and 2MWT (r = 0.633; p = 0.000). Conclusions: 2MST proved to be a reliable and valid physical performance test in patients with TKA. 2MST can be used to evaluate the physical performance of TKA patients. ClinicalTrials.gov Identifier: NCT05064943Implications for Rehabilitation The two-minute step test (2MST) is a reliable and valid test for the assessing the physical performance of patients with total knee arthroplasty (TKA). 2MST is an easy-to-administer, low-cost, and quick performance-based test that can be used routinely in any clinical setting to evaluate functional recovery after TKA. The changes of 8 or more steps for 2MST over time may represent a true change in the rehabilitation of patients with TKA.