Purpose: To establish a method for sample preparation to measure ascorbate in whole lenses and to investigate whether lens ascorbate concentration is dependent on dietary ascorbate intake. Methods: Four groups of 3 young Sprague-Dawley rats each were fed chow containing L-ascorbate, either 0.0, 5.7, 57.0 or 114.0 mmol/kg for a duration of 4 weeks. Thereafter, each rat was sacrificed. The lens was extracted, photographed, and lens wet weight was measured. The lens was homogenized in 1.0 ml of 0.25% metaphosphoric acid, the homogenate was centrifuged and the supernatant ultrafiltered. The filtrate was injected into an ion exchange, reversed-phase Polypore H HPLC column equipped with a 254-nm ultraviolet detector. Samples were calibrated against an L-ascorbate standard. Polynomial regression analysis was performed on the data. Results: All lenses were devoid of cataract. A 95% confidence interval for baseline content of ascorbate without any dietary intake was estimated to be 0.16 ± 0.01 µmol/g wet weight of lens. The lens ascorbate concentration increased linearly with dietary ascorbate intake with an increased rate, estimated as a 95% confidence interval of 0.33 ± 0.18 (µmol ascorbate) (g lens)–1 (mol ascorbate)–1 (kg chow) with r2 = 0.62. Conclusion: Lens ascorbate concentration linearly increases with dietary ascorbate intake without cataract development in the rat. The currently presented method for sample preparation to measure the whole-lens content of ascorbate is applicable.