Thiaminase I is a vitamin degrading enzyme produced by microorganisms and often found in certain metazoan animals that can cause thiamine deficiency in natural ecosystems. The physiological function of this enzyme is still unknown. We adapted a photometric assay for thiaminase I that measures the rate of disappearance of a colored co-substrate for use with a high throughput plate reader. Using this assay, we characterized the conditions that promote thiaminase I production in some thiaminase I producing microorganisms and gained evidence for and against hypotheses for various physiological functions. We also conducted surveys of diverse metazoan organisms known and not previously recognized to have thiaminase I activity and found thiaminase activity in organisms not previously known to have thiaminase activity. Research on the expression of thiaminase I has possible implications for cancer research and correction of mortality due to thiaminase-induced thiamine deficiency syndromes.