The objective of this study was to develop a spina bifida health-related quality of life (HRQOL) instrument. Items were generated through semistructured interviews, and reduced by frequency-importance product ranking. Validity was assessed by correlating the HRQOL score with a global question concerning the child's well-being using the Spearman's rank coefficient, and the Piers-Harris Children's Self-Concept Scale (P-H) using the Pearson correlation coefficient. Reproducibility was assessed at 2-week intervals using the intra-class correlation coefficient (ICC). Field testing was undertaken in a larger sample to evaluate item-total correlation, internal consistency and construct validity. Patients taking part in the study were 329 children and adolescents with spina bifida attending two treatment centres. Over 600 items were generated. These were reduced to 47 questions and 50 questions, for children and adolescents respectively. The correlation between the HRQOL score and the global question was r = 0.57, and with the P-H was 0.26 (children). These values for adolescents were 0.63, and 0.89, respectively. Reproducibility was ICC = 0.78 (children) and 0.96 (adolescents). Following field testing, the questionnaire was further reduced to 44 questions (children) and 47 questions (adolescents) by eliminating questions with an item- total correlation less than 0.20. Cronbach's alphas for the final instrument were 0.93 (children) and 0.94 (adolescents), and construct validity correlations were 0.63 (children) and 0.37 (adolescents). The spina bifida HRQOL instrument has good measurement properties and may be used as a discriminative instrument. Assessment of responsiveness is necessary before using it to evaluate therapy in clinical trials.