Fatigue in air ambulance crews leads to decrements in performance and situational awareness that may contribute to aircraft accidents and patient care mistakes. Fatigue assessments completed by flight crews can give early warning when fatigue is accumulating. Countermeasures can then be implemented to improve performance and increase safety. No validated air ambulance fatigue assessment currently exists that incorporates transport-specific factors. The objective of this study was to validate a flight fatigue assessment that accounts for air transport-specific factors. Flight crewmembers from multiple air ambulance programs participated and completed assessments. Results were analyzed to determine if the assessment captured or predicted fatigue levels of crewmembers. When used to measure crewmember fatigue, the assessment was shown to consistently and reliably confirm accumulating fatigue and correlated with crew-reported levels of fatigue. A predicted fatigue scale was created to help crewmembers objectively identify their fatigue level. Used consistently, the transport fatigue assessment should increase awareness of accumulating fatigue. With awareness, crewmembers are better equipped to justify and take advantage of opportunities to mitigate their fatigue, increase crew coordination, enhance safety, and improve patient care.