The objective of the present study is to propose an experimental technique to assess the effect of glove stretch on the barrier performance of a variety of intact medical glove materials. Methods: We improved a consensus method by incorporating domed shaped screens into the penetration cell, which enhances the detection of failures for glove integrity. Two screen types and three glove materials were selected to compare the barrier performance of unstretched and stretched glove specimens and the performance of various glove materials. Fluorometric analysis was used to detect fluid challenge with fluorescent dye in the recovery buffer to indicate barrier failure. Results: In the visual inspection method, 5 sample failures were found (5.68%; n=88) regardless of test condition. All three glove materials provided similar results and were not significantly different. The differences in failure rates between stretch and unstretched gloves were insignificant. By dividing the detected fluorescent concentrations into groups: above LOQ, between LOD and LOQ, the domed-shape screen provided greater sensitivity to assess glove barrier performance between the LOQ and LOD when glove was in a stretched state (18.18%) compared to the unstretched state (2.27%). The fluorometric method demonstrated a better detection sensitivity (AUC=0.848) compared to visual inspection method (AUC= 0.608). Conclusions: The present study confirms not only previous observations that nitrile examination gloves are a suitable alternative to the latex but also adds to the literature showing the use of a fluorescence agent and a fluorometric spectroscopy may provide an improved detection mechanism compared to visual penetration.