Background <p>Fragile X Syndrome (FXS) is a rare X-linked genetic condition. Children with FXS exhibit a variety of challenging behaviors such as irritability, anxiety, and preference for solitary activities. The Aberrant Behavior Checklist-Community (ABC-C) is an observer-reported outcome (ObsRO) measure that captures many of the behavioral concerns (e.g., outbursts, social anxiety, difficulty connecting with others) salient to caregivers of individuals with FXS. An FXS-specific scoring algorithm of the ABC-C, considered more representative of the FXS phenotype (ABC-C_FXS), has been included in numerous clinical studies of treatments for FXS. The goal of this qualitative research was to establish the content validity of the ABC-C_FXS domain structure in parents/caregivers of children with FXS and determine whether it is fit-for-purpose for measuring treatment benefit in clinical studies of FXS.</p> Methods <p>The qualitative data came from 3 sources: (1) Concept elicitation (CE) interviews with caregivers of children with FXS (<i>N</i> = 10); (2) Caregiver survey (CS) (<i>N</i> = 212) completed at entry to CONNECT-FX (NCT03614663); and (3) Cognitive interviews of the ABC-C_FXS with caregivers of children with FXS (<i>N</i> = 25). Qualitative coding and analysis were consistent with best practices and followed principles in applied thematic (content) analysis. Item comprehension and relevancy of the ABC-C_FXS were also examined based on data from cognitive interviews.</p> Results <p>The mean age of individuals with FXS was 10 (range 3–17) years and was consistent across the 3 data sources. The most frequently reported behavioral challenges described by caregivers were generally consistent across the CE interviews and the CS. Socially avoidant behaviors were most prevalent (70% and 49%, respectively) followed by socially unresponsive/lack of interaction (60% and 51%, respectively) and aggression (60% and 43%, respectively). Saturation of behavioral challenges related to FXS was reached in both the CE interviews and CS and was consistent with the content of the ABC-C_FXS. Cognitive interviews showed that caregivers could understand and interpret the content and response options of the ABC-C_FXS and deemed its content relevant to their experience.</p> Conclusions <p>The content validity evidence from this qualitative research supports the use of ABC-C_FXS as an efficacy measure in FXS clinical research studies.</p> Clinical trial registration <p>Not applicable.</p>