Introduction and ObjectiveThe cold chain of immunobiological agent conservation occupies a strategic position in the immunization system and, therefore, needs to be evaluated. This study psychometrically evaluated the Immunobiological Agent Conservation Assessment Scale (Escala de Avaliação da Conservação de Imunobiológicos—EACI).MethodsMethodological study carried out in Minas Gerais, Brazil, including 275 immunization rooms, divided into three stages: (a) pilot study; (b) internal consistency and temporal reproducibility; (c) criterion validity and structural validity.ResultsThe EACI items were analyzed for comprehension and clarity; presenting internally consistency (Cronbach’s alpha: 0.72 [95% CI: 0.666–0.763]) and temporal reproducibility (ICC: 0.948 [95% CI: 0.911–0.981]), in addition to explaining 72% of the variance and discriminating the groups criteria (p = .0025).ConclusionThe EACI is psychometrically reliable and valid and is the first assessment instrument available for this construct.