Glyphosate and glufosinate are widely used herbicides and, thus, there is an interest in the reliable and sensitive determination of glyphosate in water and food. These pesticides are difficult to extract and analyze because of their high polarity. Here we describe an automated workflow for the FMOCderivatization, sample cleanup, and LC-MS/MS detection using a GERSTEL Multi Purpose Sampler (MPS) 2XL configured with an online solid phase extraction (SPEXOS) module coupled to an AB SCIEX QTRAP� 4500 system for the identification and quantitation of glyphosate, its major metabolite AMPA, and glufosinate in water and food samples.