Detection and analysis of bacteria from environmental samples (e.g. water, air, and food) are usually accomplished by standard culture techniques or by analyses that target specific DNA sequences, antigens or chemicals. For large cell numbers in aqueous suspensions, an alternative technique that has proven useful is total luminescence spectroscopy (TLS). TLS is the acquisition of fluorescence data that records the unique excitation-emission matrix (EEM) of compound fluorophores. Past work has shown that one type of bacterial endospore, Bacillus megaterium, possessed a distinct EEM pattern useful for differentiating it in complex biological fluids and suspensions. The work described here extends those observations to establish some limits on the sensitivity and specificity of TLS for the detection and analysis of bacterial endospores versus (bacterial) vegetative cells in aqueous culture. Our findings show Bacillus endospores exhibit a dramatic blue shift of 130 nm in excitation and a smaller shift of 50 nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.