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<b>Aims:</b> The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon&#x2032;s tissue following application over the intact conjunctiva before routine trabeculectomy. <b> Settings and Design:</b> Institution-based case series. <b> Materials and Methods:</b> In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon&#x2032;s capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC). <b> Statistical Analysis Used:</b> Statistical analysis was performed using stata0 8.0 version software (STATA Corporation, Houston, TX, USA). In this study, <i> P</i> -values less than 0.05 were considered as statistically significant. <b> Results:</b> The average weight of the sample of Tenon&#x2032;s tissue excised was 5.51 &#x00B1; 4.42 mg (range: 0.9-17.1) and the average estimated MMC concentration found to be present in Tenon&#x2032;s tissue using HPLC was 18.67 &#x00B1; 32.36 x 10^&#8722;6 moles/kg of the tissue (range: 0.38-197.05 x 10^&#8722;6 ). In 36 of the 41 patients (87.80&#x0025;), the MMC concentration reached above 2 x 10^&#8722;6 moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. <b> Conclusions:</b> Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.