Background.Tear ascorbate is important for corneal health. A rapid and simple method for measurement of ascorbate in tears is needed, and adequate knowledge of physiological variation of tear ascorbate is important to facilitate comparative studies of the effect of, for example, contact lens wear and environmental conditions and stresses. However, there are currently no data on physiological variation of tear ascorbate. This study validated a simple and speedy method for tear ascorbate and investigated between-eye and between-day variation in tear ascorbate in healthy young adults.Methods.Yawn-induced reflex tears were collected from 32 healthy Hong Kong Chinese subjects and measured by both high-performance liquid chromatography (HPLC) and by an enzyme-linked colorimetric method known as FRASC (total ferric reducing (antioxidant) activity and ascorbate concentration measurement). For between-eye variation, yawn reflex tears were collected from each eye of the same 32 healthy subjects, and ascorbate was measured using HPLC; in a separate experiment for between-day variation, tears were collected on two separate days from 14 subjects, and ascorbate was measured by FRASC.Results.Both HPLC and FRASC showed high precision, and results obtained using FRASC were not statistically different from those using HPLC; mean ± SD were, respectively, 18.5 ± 4.4 &mgr;M and 18.5 ± 4.8 &mgr;M for HPLC and FRASC methods (p = 0.943). No significant between-eye difference in tear ascorbate was found (p = 0.386), and no significant between-day variation was found overall: mean ± SD ascorbate was 20.0 ± 6.2 &mgr;M on day 1 and 19.3 ± 6.8 &mgr;M on day 2 (p = 0.772). However, between-day variation was large in seven of 14 subjects.Conclusion.FRASC is an acceptable alternative to HPLC for measurement of tear ascorbate. Tears for ascorbate investigation can be collected from either eye or, if necessary, from both eyes and pooled. However, tear ascorbate may vary widely from day to day in the same individual. The reasons for this variation require further study but may relate to differences in ascorbate supply or demand within the precorneal tear layer.