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Background: Failing to account for the resources required to successfully implement public health interventions can lead to an underestimation of costs and budget impact, optimistic cost-effectiveness estimates, and ultimately a disconnect between published evidence and public health decision-making. Methods: We developed a conceptual framework for assessing implementation costs. We illustrate the use of this framework with case studies involving interventions for tuberculosis and HIV/AIDS in resource-limited settings. Results: Costs of implementing public health interventions may be conceptualized as occurring across three phases: design, initiation, and maintenance. In the design phase, activities include developing intervention components and establishing necessary infrastructure (e.g., technology, standard operating procedures). Initiation phase activities include training, initiation of supply chains and quality assurance procedures, and installation of equipment. Implementation costs in the maintenance phase include ongoing technical support, monitoring and evaluation, and troubleshooting unexpected obstacles. Within each phase, implementation costs can be incurred at the site of delivery (“site-specific” costs) or more centrally (“above-service” or “central” costs). For interventions evaluated in the context of research studies, implementation costs should be classified as programmatic, researchrelated, or shared research/program costs. Purely research-related costs are often excluded from analysis of programmatic implementation. Conclusions: In evaluating public health interventions in resource-limited settings, accounting for implementation costs enables more realistic estimates of budget impact and cost-effectiveness and provides important insights into program feasibility, scale-up, and sustainability. Assessment of implementation costs should be planned prospectively and performed in a standardized manner to ensure generalizability.