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Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium‐enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope‐ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C₁₆, C₁₈ acids and leaf waxes (C₂₃–C₃₁ n‐alkanes; C₂₂–C₃₀ n‐acids) over a diurnal …