Background: Prolonged sitting time has been identified as a health risk factor. Sit-stand workstations allow desk workers to alternate between sitting and standing throughout the working day, but not much is known about their acceptability and feasibility. Hence, the aim of this study was to qualitatively evaluate the acceptability, feasibility and perceptions of using sit-stand workstations in a group of desk-based office workers. Methods: This article describes the qualitative evaluation of the randomized controlled cross-over Stand@Work pilot trial. Participants were adult employees recruited from a non-government health agency in Sydney, Australia. The intervention involved using an Ergotron Workfit S sit-stand workstation for four weeks. After the four week intervention, participants shared their perceptions and experiences of using the sit-stand workstation in focus group interviews with 4-5 participants. Topics covered in the focus groups included patterns of workstation use, barriers and facilitators to standing while working, effects on work performance, physical impacts, and feasibility in the office. Focus group field notes and transcripts were analysed in an iterative process during and after the data collection period to identify the main concepts and themes. Results: During nine 45-min focus groups, a total of 42 participants were interviewed. Participants were largely intrinsically motivated to try the sit-stand workstation, mostly because of curiosity to try something new, interest in potential health benefits, and the relevance to the participant's own and organisation's work. Most participants used the sit-stand workstation and three common usage patterns were identified: task-based routine, time-based routine, and no particular routine. Common barriers to sit-stand workstation use were working in an open plan office, and issues with sit-stand workstation design. Common facilitators of sit-stand workstation use were a supportive work environment conducive to standing, perceived physical health benefits, and perceived work benefits. When prompted, most participants indicated they were interested in using a sit-stand workstation in the future. Conclusions: The use of a sit-stand workstation in this group of desk-based office workers was generally perceived as acceptable and feasible. Future studies are needed to explore this in different desk-based work populations and settings.