A sensitive, automated, non-competitive fluorescent immunoassay was developed for quantitative analysis of 3-phenoxybenzoic acid (PBA) in human urine sampes as a putative biomarker of exposure to pyrethroid insecticides using the bench top immunoanalyzer, KinExA™ 3000 system. The key difference between the KinExA system and the enzymelinked immunosorbent assay (ELISA) is to eliminate the PBA-antibody interaction with the coating antigen. This can be achieved by separately capturing the free PBA-antibody onto the hapten-immobilized beads when a constant amount of reaction solution in equilibrium between PBA-antibody and analyte passes through the bead-packed glass capillary column. Optimal dilution of the PBA antibody was determined when fluorescent signals of 0.5-2 were obtained and a sufficient amount of coating antigen was immobilized to ensure the capture of all free antibodies. IC 50S of the two KinExA methods (0.3 and 0.6 ng/mL for one- and two-step KinExA, respectively) were 3- and 6-fold better than the heterologous ELISA and were approximately 650- and 300-fold lower compared to that of the homologous ELISA (IC 50 of 200 ng/mL). The KinExA assay was negligibly affected within tested range of pHs (5-10) and ionic strengths (1, 5, and 10X PBS). Similar urine matrix effects were observed in the two KinExA assays with a 5- to 10-fold increase in IC 50s when 5 and 10% of urine was contained in the reaction buffer. A high correlation (r 2 = 0.99) was observed between detected and spiked concentrations of PBA standard with average recoveries of 88-160%.