Whilst the methodology of adult letter acuity measurement has been substantially refined over the last two decades, relatively little development has occurred in methods for quantifying letter acuity in young children. This study compares a recently developed visual acuity test (Glasgow Acuity Cards), which incorporates several key design features used in adult test charts to improve the sensitivity and reliability of visual acuity measurements. The equivalence of acuity measurements made with Glasgow Acuity Cards were compared with the Bailey-Lovie logMAR chart and Snellen chart in adults, and with traditional Single Letter Acuity and a modified Single Letter Acuity test in children. The test-retest reliability of acuity measurements made with Glasgow Acuity Cards and the Single Letter Acuity tests were also assessed in a large group of visually normal children. In addition, the ability of the pre-school letter acuity tests to detect differences in acuity between the two eyes, and to detect amblyopia were examined. Ninety-five percent of vision measurements made with the Bailey-Lovie chart and Glasgow Acuity Cards differ by less than 0.07 log unit. Furthermore, the sensitivity of Glasgow Acuity Cards to detecting changes in acuity longitudinally and inter-ocular differences in acuity is considerably greater as compared with traditional Single Letter Acuity tests. Improvements in paediatric acuity chart design are important for the effective detection and management of children with amblyopia.