Nosocomial infections (NIs) are among the main serious adverse events in health care that are preventable. The Québec Ministry of Health and Social Services (MSSS) set forth a 2015-2020 action plan to evaluate the costs and assess the return on investment of NI prevention and control (NIPC) programs. Some studies related to the costs of clinical best practices (CBPs) associated with NIPC (hand hygiene, hygiene and sanitation, screening of carriers and infected patients, and application of basic and additional precautions) exist in the scientific literature. However, to the best of our knowledge, no standardized instrument currently exists that effectively captures the costs of these CBPs. Objective: To fill this gap, this study sought to develop, validate and pilot test a time-motion grid to assess the costs of these four CBPs. We reviewed the literature on the costs of control and prevention of NIs. An algorithm and time-motion grid were developed and validated using the Delphi approach, over two rounds, by 18 clinical experts and professionals in the field of NIPC. A pilot test realized over a two week period in medical and surgical units of two hospitals involved 48 staff members: nurses, nursing assistants, orderlies and hygiene and sanitation staff. Staff time required to complete a CBP, as well as materials and cleaning products used, were also simultaneously collected. Conclusion: The final grid was adapted to a mobile platform. It is a useful and validated instrument that estimates the costs of CBPs and may help optimize NIPC programs.