Background: According to the World Bank, Malaysia reported an estimated 97 tuberculosis cases per 100,000 people in 2021. Chest x-ray (CXR) remains the best conventional method for the early detection of pulmonary tuberculosis (PTB) infection. The intervention of artificial intelligence (AI) in PTB diagnosis could efficiently aid human interpreters and reduce health professionals’ work burden. To date, no AI studies have been evaluated in Malaysia. Objective: This study aims to evaluate the performance of Putralytica and Qure.ai software for CXR screening and PTB diagnosis among the Malaysian population. Methods: We will conduct a retrospective case-control study at the Respiratory Medicine Institute, National Cancer Institute, and Sungai Buloh Health Clinic. A total of 1500 CXR images of patients who completed treatments or check-ups will be selected and categorized into three groups: (1) abnormal PTB cases, (2) abnormal non-PTB cases, and (3) normal cases. These CXR images, along with their clinical findings, will be the reference standard in this study. All patient data, including sociodemographic characteristics and clinical history, will be collected prior to screening via Putralytica and Qure.ai software and readers’ interpretation, which are the index tests for this study. Interpretation from all 3 index tests will be compared with the reference standard, and significant statistical analysis will be computed. Results: Data collection is expected to commence in August 2023. It is anticipated that 1 year will be needed to conduct the study. Conclusions: This study will measure the accuracy of Putralytica and Qure.ai software and whether their findings will concur with readers’ interpretation and the reference standard, thus providing evidence toward the effectiveness of implementing AI in the medical setting. International Registered Report Identifier (IRRID): PRR1-10.2196/36121