With over a quarter of the world’s bats species facing extinction, there is a need for ecotoxicological studies to assess if acute and sublethal exposure to newer pesticides such as neonicotinoids and carbonates contribute to population declines. Pesticide exposure studies in bats have been limited to terminal sampling methods, therefore we developed a non-invasive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing hair trimmings. The hair of big brown bats (Eptesicus fuscus) was collected and pooled by county to assess the best extraction solvent and solid-phase-extraction (SPE) clean-up cartridges. Using the best performing extraction solvent, methanol, and the best performing SPE cartridge, Chromabond HR-X, we developed an optimized multiple reaction monitoring (MRM) LC-MS/MS method for simultaneous determination of 3 neonicotinoids, clothianidin, imidacloprid, and thiamethoxam; 1 carbonate, carbaryl; and 4 systemic herbicides, 2,4-D, atrazine, dicamba, and glyphosate. The optimized protocol yielded the detection of 3–8 of the compounds in the county-level bat hair pools. 2,4-D, glyphosate, and imidacloprid were found in all samples with two of the county-level hair samples having glyphosate concentrations of over 3500 pg/mg of hair. This approach has great potential to facilitate non-terminal ecotoxicological studies assessing the effects of subacute (chronic) pesticide exposure in threatened and endangered bat species and other species experiencing population declines.