ABSTRACT The increased demand for sterile products has created the need for rapid technologies capable of validating the hygiene of industrial production processes. Bacillus endospores are in standard use as biological indicators for evaluating the effectiveness of sterilization processes. Currently, culture-based methods, requiring more than 2 days before results become available, are employed to verify endospore inactivation. We describe a rapid, microscopy-based endospore viability assay (μEVA) capable of enumerating germinable endospores in less than 15 min. μEVA employs time-gated luminescence microscopy to enumerate single germinable endospores via terbium-dipicolinate (Tb-DPA) luminescence, which is triggered under UV excitation as 10 8 DPA molecules are released during germination on agarose containing Tb 3+ and a germinant (e.g., l -alanine). Inactivation of endospore populations to sterility was monitored with μEVA as a function of thermal and UV dosage. A comparison of culturing results yielded nearly identical decimal reduction values, thus validating μEVA as a rapid biodosimetry method for monitoring sterilization processes. The simple Tb-DPA chemical test for germinability is envisioned to enable fully automated instrumentation for in-line monitoring of hygiene in industrial production processes.