Background Since desk-dominated work environments facilitate sedentary behavior, office workers sit for 66% of their working days and only 8% succeed in interrupting their prolonged periods of sitting within the first 55 minutes. Yet stretches of long and uninterrupted sitting increase the likelihood of several chronic metabolic and cardiovascular diseases. Objective We therefore developed a computer-based app designed to interrupt periods of prolonged sitting among office employees. Methods When developing the intervention, we applied the intervention mapping protocol. This approach for the systematic design of theory and evidence-based behavior change programs consists of 6 steps: creation of a logic model of the problem, creation of a logic model of change, program design, program production, design of an implementation plan, and development of an evaluation plan. Results Working through all 6 steps has resulted in an individually adaptable intervention to reduce sedentary behavior at work. The intervention, UPcomplish, consists of tailored, half-automatized motivational components delivered by a coach. To register sedentary behavior, the VitaBit (VitaBit Software International BV) toolkit, a wearable accelerometry-based monitoring device, is used. Among others, UPcomplish includes personalized goal setting, tailored suggestions to overcome hurdles, and weekly challenges. The VitaBit toolkit supports the participants to monitor their behavior in relation to self-set goals. Conclusions Intervention mapping is a useful protocol not only for the systematic development of a comprehensive intervention to reduce sedentary behavior but also for planning program adherence, program implementation, and program maintenance. It facilitates obtaining the participation of relevant stakeholders at different ecological levels in the development process of the intervention and anticipating facilitators to and barriers of program implementation and maintenance. Trial Registration Netherlands Trial Register NL7503; https://www.trialregister.nl/trial/7503