The biocontainment unit at Johns Hopkins Hospital is a specially designed, inactive high-level isolation unit designated to care for patients infected with high-consequence pathogens. The unit team designed a facility-specific readiness scale and checklist that focus on infrastructure, consumable supplies, and staffing to assess activation readiness of the biocontainment unit. Over a period of 50 days and 14 days, these tools were used as part of a routine risk assessment to first identify barriers and then tier the impact of these barriers into activation categories of "Ready," "Ready with Considerations," and "Not Ready." The assessment identified the greatest risks to activation readiness were staffing and waste management capabilities. Assessing threats to activation readiness and the risk of not being ready should be a priority for maintaining facility, regional, and national capacity to safely isolate and care for patients infected with high-consequence pathogens while maintaining healthcare worker safety.