Glyphosate is the most widely used agricultural herbicide in the world and in the United States. In this study, we measured glyphosate levels in water, foods, and human urine samples. We also specifically examined possible correlations between urinary glyphosate levels and physiological, dietary, or behavioral factors. The results show that water samples obtained from both Lake Erie and tap water contained nondetectable or very low levels of glyphosate (≤0.08 ppb). On the other hand, food samples showed various levels of glyphosate contamination. In particular, tea products (40–728 ppb in five samples), coffee powder (11 and 26 ppb in two samples), and honey (20 ppb) had higher glyphosate concentrations in comparison to other foods. Glyphosate was also detected in human urine samples, in particular at elevated levels (~2 fold) from participants who consumed tea in the past 24 h.