During July 2016-January 2017, two unrelated measles cases were identified in the Denver, Colorado area after patients traveled to countries with endemic measles transmission. Each case resulted in multiple exposures at health care facilities and public venues, and activated an immediate and complex response by local and state public health agencies, with activities led by the Tri-County Health Department (TCHD), which serves Adams, Arapahoe, and Douglas counties. To track the economic burden associated with investigating and responding to single measles cases, personnel hours and supply costs incurred during each investigation were tracked prospectively. No secondary cases of measles were identified in either investigation. Postexposure prophylaxis (PEP) was administered to 31 contacts involving the first case; no contacts of the second case were eligible for PEP because of a delay in diagnosing measles disease. Public health costs of disease investigation in the first and second case were estimated at $49,769 and $18,423, respectively. Single measles cases prompted coordinated public health action and were costly and resource-intensive for local public health agencies.