Background: The goal of user-centered design (UCD) is to understand the users’ perspective and to use that knowledge to shape more effective solutions. The UCD approach provides insight into users’ needs and requirements and thereby improves the design of the developed services. However, involving users in the development process does not guarantee that feedback from different subgroups of users will shape the development in ways that will make the solutions more useful for the entire target user population. Objective: The aim of this study was to describe a protocol for systematic analysis and prioritization of feedback from user subgroups in the usability testing of a digital motivation support for fall-preventive physical activity (PA) interventions in seniors (aged 65 years and older). This protocol can help researchers and developers to systematically exploit feedback from relevant user subgroups in UCD. Methods: Gender, PA level, and level of technology experience have been identified in the literature to influence users’ experience and use of digital support systems for fall-preventive PA interventions in seniors. These 3 key user characteristics were dichotomized and used to define 8 (ie, 23) possible user subgroups. The presented method enables systematic tracking of the user subgroups’ contributions in iterative development. The method comprises (1) compilation of difficulties and deficiencies in the digital applications identified in usability testing, (2) clustering of the identified difficulties and deficiencies, and (3) prioritization of deficiencies to be rectified. Tracking user subgroup representation in the user feedback ensures that the development process is prioritized according to the needs of different subgroups. Mainly qualitative data collection methods are used. Results: A protocol was developed to ensure that feedback from users representing all possible variants of 3 selected key user characteristics (gender, PA level, and level of technology experience) is considered in the iterative usability testing of a digital support for seniors’ PA. The method was applied in iterative usability testing of two digital applications during spring/summer 2018. Results from the study on the users’ experiences and the iterative modification of the digital applications are expected to be published during 2021. Conclusions: Methods for systematic collection, analysis, and prioritization of feedback from user subgroups might be particularly important in heterogenous user groups (eg, seniors). This study can contribute to identifying and improving the understanding of potential differences between user subgroups of seniors in their use and experiences of digital support for fall-preventive PA interventions. This knowledge may be relevant for developing digital support systems that are appropriate, useful, and attractive to users and for enabling the design of digital support systems that target specific user subgroups (ie, tailoring of the support). The protocol needs to be further used and investigated in order to validate its potential value.