Needlestick injuries that expose workers to infection by blood-borne pathogens can be prevented by the use of appropriate protective gloves. Recently, an F23 ASTM task group developed the test method ASTM F2878-10, which defines needle puncture resistance as the maximum force recorded during tests using one of three hypodermic needle gauges. This study has two objectives: (1) to assess the effect of needle gauge on the ranking of glove puncture performance using the ASTM F2878-10 standard test method, and (2) to analyze the correspondence between maximum puncture force and puncture-through using an electrical detection device during needle penetration in protective gloves made of reinforced and multilayer composite materials. Two series of experiments were performed. In the first one, seven glove materials were characterized as described in the ASTM test method. The glove puncture resistance results were ranked after testing with the 21, 25, and 28 needle gauges. In the second experiment, a test device was used to electrically detect the force at which the needle tip punctures the material. Puncture tests were performed on seven glove materials following the ASTM F2878-10 test method and using this electrical device. The maximum force and force at puncture-through criteria are discussed. In the first experiment, the needle puncture resistance ranking of the gloves was almost equivalent for all three needle gauges. For glove selection purposes, the use of a single needle gauge provides sufficient information on needle puncture resistance for a comparison of the performance of the protective glove materials. In the second experiment, for two of the seven glove models, needle puncture-through was detected before the maximum force was reached. For those cases, the force at puncture-through criterion provides additional information on the material resistance that is not fully captured by the maximum force criterion alone, but it requires a more complex experimental setup.