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Background: Despite the increasing number of mobile health applications, the validity of their content is understudied. The objective of this study was to rate the content of HIV/AIDS-related mobile applications and to determine the extent to which evidence-based medicine is being incorporated into their content using a new tool called the Evidence-based content rating tool of mobile health applications (EBCRT-mHealth).Methods: All available HIV/AIDS-related applications in Iran from Cafe Bazaar and Google Play Store were evaluated. This study was first conducted in 2018, then after almost two years in 2021 was done again. In this study, researchers developed the EBCRT-mHealth tool to rate the content of applications based on the evidence-based medicine pyramid. Its reliability was calculated (α = 0.78), and five specialists confirmed its validity. Two reviewers independently reviewed all HIV/AIDS applications directly downloaded and installed from the Google Play Store and Cafe Bazaar.Results: Out of 980 retrieved applications, in 2018, 85, and in 2021, 78 applications were included in the study. Only in 17 (28%) out of the 60 in 2018, and 25 (51%) in 2021 Google Play store applications the source of content information was mentioned. All Cafe Bazaar mobile applications mentioned the source of information. The mean rating of all application content in 2018 was 2.38 (SD = 0.74), and in 2021 was 2.90 (SD = 1.35) out of 5. The mean rating of the content of Cafe Bazaar applications in 2018 was 2.10 (SD = 0.49), and in 2021 was 1.94 (SD = 0.29). The mean content rating of Google Play store applications in 2018 was 2.50 (SD = 0.80) and in 2021 was 3.86 (SD = 1.18).Conclusion: After two years, the rating of the content of HIV/AIDS-related applications available in Iran that existed in Cafe Bazaar decreased from "poor" to "inappropriate". Also, the content score of the Google Play Store applications increased from "poor" to "good". It is critical to ensure the credibility of the sources used in developing their content and removing applications with inappropriate and unreliable content from the App Stores. Also, mobile health application developers should use the highest quality information in their applications. [ABSTRACT FROM AUTHOR]