The Automated Self-Administered Dietary Assessment Tool (ASA24) is a web-based tool that guides participants through completion of a 24-h dietary recall and automatically codes the data. Despite the advantages of automation, eliminating interviewer contact may diminish data quality. Usability testing can assess the extent to which individuals can use the ASA24 to report dietary intake with efficiency, effectiveness, and satisfaction. This mixed-methods study evaluated the usability of the ASA24 to quantify user performance and to examine qualitatively usability issues in a sample of low-income adults (85% female, 48.2 years on average) participating in a nutrition coupon program. Thirty-nine participants completed a 24-h dietary recall using the ASA24. Audio and screen recordings, and survey responses were analyzed to calculate task times, success rates, and usability issue frequency. Qualitative data were analyzed thematically to characterize usability issues. Only one participant was able to complete a dietary recall unassisted. We identified 286 usability issues within 22 general usability categories, including difficulties using the search function, misunderstanding questions, and uncertainty regarding how to proceed to the next step; 71.4% of participants knowingly misentered dietary information at least once. Usability issues may diminish participation rates and compromise the quality of ASA24 dietary intake data. Researchers should provide on-demand technical support and designers should improve the intelligence and flexibility of the ASA24’s search functionality.