S(−)-Satropane is currently being developed to in situ forming ophthalmic gel, a new ophthalmic delivery system, for the treatment of glaucoma. To evaluate the pharmacokinetic profiles of S(−)-satropane, the microdialysis method was employed. The concentration of S(−)-satropane in dialysates was measured by using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Unlike the common solution prepared in normal saline, in which the level of S(−)-satropane in aqueous humor increased rapidly after instillation and reached the maximal level (C max of 1.508±0.297μgml −1 ) within 1h, S(−)-satropane exhibited 3.2-fold greater C max and 2.2-fold greater AUC 0–3h (p<0.05) in the in situ forming gel. The results showed that the in situ forming gel system could improve the ocular bioavailability of S(−)-satropane.