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Background: Standardized and validated heat inactivation procedure for Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not available. For heat inactivation, various protocols were reported to prepare External Quality Assessment Programme (EQAP) samples without direct comparison between different durations., Objective: To assess the heat inactivation procedures against SARS-CoV-2. The efficacy of the optimized condition was reflected by the results from laboratories testing the EQAP samples., Study Design: The SARS-CoV-2 strain was exposed to 95 °C in a water bath for three different time intervals, 5 min, 10 min and 15 min, respectively. The efficacy of inactivation was confirmed by the absence of cytopathic effects and decreasing viral load in 3 successive cell line passages. The viral stock inactivated by the optimal time interval was dispatched to EQAP participants and the result returned were analyzed., Results: All of the three conditions were capable of inactivating the SARS-CoV-2 of viral load at around cycle threshold value of 10. When the 95 °C 10 min condition was chosen to prepare SARS-CoV-2 EQAP samples, they showed sufficient homogeneity and stability. High degree of consensus was observed among EQAP participants in all samples dispatched., Conclusions: The conditions evaluated in the present study could be helpful for laboratories in preparing SARS-CoV-2 EQAP samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)