Polyethoxylated tallow amine (POEA) surfactants in glyphosate formulations are understudied. They may constitute greater health risks than glyphosate itself. Lack of validated biomarkers of exposure and metabolism, as well as analytical methods for measuring POEA, limit study of a formulation's toxicity and associated risk. We report an exploratory analytical workflow using liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-MS/MS) in conjunction with neutral loss (NL) and product ion (PI) screening tools as an effective data acquisition tool to screen for POEA homologues in human urine. These features made it possible to find POEA at lower levels, which would not have been possible without screening tools and chemical standards.