Background: Despite international efforts to protect and promote exclusive breastfeeding (EBF) for infants up to six months of age, global rates of EBF continue to fall short of the targets proposed by the WHO for 2025. Previous studies have shown a relationship between the level of health literacy and the duration of EBF, although this relationship was not determinant, probably due to the use of a generic health literacy questionnaire. Therefore, this study aims to design and validate the first specific breastfeeding literacy instrument. Methods: A Breastfeeding Literacy instrument was developed. Content validation was carried out by a group of 10 experts in health literacy, breastfeeding or instrument validation, obtaining a Content Validity index in Scale (S-CVI/Ave) of 0.912. A multicentre cross-sectional study was carried out in three Spanish hospitals to determine the psychometric properties (construct validity and internal consistency). The questionnaire was administered to 204 women during the clinical puerperium. Results: The Kaiser-Meier-Oklin Test (KMO = 0.924) and Bartlett’s Test of Sphericity (X2 = 3119.861; p ≤ 0.001) confirmed the feasibility of the Exploratory Factor Analysis, which explained 60.54% of the variance with four factors. Conclusions: The Breastfeeding Literacy Assessment Instrument (BLAI) consisting of 26 items was validated.