<p>Assessing usability and satisfaction is vital to ensure the efficiency and optimal use of mobile health (mHealth) applications. Nevertheless, existing questionnaires revolve around computerized systems and lack validation for evaluating mHealth applications. We aimed to develop and validate a tri-language questionnaire to assess usability and satisfaction of mobile health applications (USHA). This study consisted of three phases: item development, translation, and validation. During the item development phase, a preliminary English version of the USHA questionnaire that comprised Likert-scale and demographic items was designed. Subsequently, forward-backward translation was performed to produce Malay and Chinese versions. Content validation was conducted with eight experts, followed by face validation with five diabetes mellitus patients. Reliability testing was conducted through test-retest analysis among diabetes mellitus patients. The initial tri-language USHA questionnaire consisted of 18 Likert-scale items and 8 demographic items. Following expert validation, five Likert-scale items and one demographic item were eliminated for lack of relevance, importance, or clarity, while four Likert-scale items were rephrased. During face validation, additional one demographic item was removed. The finalized questionnaire demonstrated high reliability, with a Cronbach’s alpha of 0.956 and an intraclass correlation coefficient of 0.845. Consequently, the tri-language USHA questionnaire consisted of 13 Likert-scale items and six demographic items, is a valid and reliable instrument that enhances accessibility and enables assessment of the usability and satisfaction of interactive mHealth applications, especially for diabetes mellitus care across a broad range of users.</p>