Lack of reliable methods to accurately measure hydrogen sulfide (H 2 S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H 2 S in cell culture media following incubation with H 2 S-releasing compounds. However, this method is inaccurate because H 2 S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H 2 S, allowing measurement of H 2 S accumulated with time. The amount of H 2 S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H 2 S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10μM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50μM NaHS, we were able to recover twice more H 2 S than conventional methods. When H 2 S-releasing compounds l-cysteine and N-acetylcysteine were added to the cell culture, the amount of H 2 S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H 2 S generated in vitro. This method could be used to screen compounds to identify potential H 2 S donors and inhibitors for therapeutic use.