Glufosinate is a widely used herbicide, which is difficult to detect by conventional analytical techniques. For many other herbicides, suitable antibodies have been raised for immunoassay development. Unfortunately, glufosinate is a very small molecule and difficult to immunize with. Thus, a derivatization-assisted immunoassay (DAIA) using the target analyte N-acetyl-glufosinate (NAG) was constructed. The activated hapten was synthesized by a new approach, using a homobifunctional cross-linker suberic acid bis(N-hydroxysuccinimide ester). The preparation of a suitable conjugate, the immunization, and the characterization of polyclonal antibodies are shown. The determination of the conjugation density (hapten density) of the immunogens was performed by four different methods (high-performance liquid chromatography with a refractive index detector, total reflection X-ray fluorescence, inductively coupled plasma mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), which gave similar results. The limit of detection was 17 mug/L NAG in water for the direct competitive enzyme immunoassay. NAG is also a main metabolite of glufosinate in resistant transgenic plants. The antibodies might be useful for the selective detection of NAG in the presence of the parent compound glufosinate (cross-reactivity 0.13%) and other metabolites.