Background: The workplace is a prominent domain for excessive sitting. The consequences of increased sitting time include adverse health outcomes such as cardiovascular disease and poor mental wellbeing. There is evidence that breaking up sitting could improve health, however, any such intervention in the workplace would need to be informed by a theoretical evidence-based framework. The aim of this study was to use the Behaviour Change Wheel (BCW) to develop a tailored intervention to break up and reduce workplace sitting in desk-based workers. Methods: The BCW guide was followed for this qualitative, pre-intervention development study. Semi-structured interviews were conducted with 25 office workers (26-59 years, mean age 40.9 [SD=10.8] years; 68% female) who were purposively recruited from local council offices and a university in the East of England region. The interview questions were developed using the Theoretical Domains Framework (TDF). Transcripts were deductively analysed using the COM-B (Capability, Opportunity, Motivation – Behaviour) model of behaviour. The Behaviour Change Technique Taxonomy Version 1 (BCTv1) was thereafter used to identify possible strategies that could be used to facilitate change in sitting behaviour of office workers in a future intervention. Results: Qualitative analysis using COM-B identified that participants felt that they had the physical Capability to break up their sitting time, however, some lacked the psychological Capability in relation to the knowledge of both guidelines for sitting time and the consequences of excess sitting. Social and physical Opportunity was identified as important, such as a supportive organisational culture (social) and the need for environmental resources (physical). Reflective and automatic Motivation was highlighted as a core target for intervention. Seven intervention functions and three policy categories from the BCW were identified as relevant. Finally, 39 behaviour change techniques (BCTs) were identified as potential active components for an intervention to break up sitting time in the workplace. Conclusions: The TDF, COM-B model and BCW can be successfully applied through a systematic process to understand the drivers of behaviour of office workers to develop a co-created intervention that can be used to break up and decrease prolonged sitting in the workplace.