A solid-phase microextraction (SPME) protocol was developed for the quantitative analysis of monocyclic aromatic amines from biological fluids. The headspace SPME sampling technique was optimized for extraction and concentration of five target analytes (aniline, o-toluidine, 2-chloroaniline, 2,6-dimethylaniline, 2,4,6-trimethylaniline) from urine, blood, and milk. The temperature, pH, and ionic strength of the matrix sample were modified to allow maximum adsorption of the analytes onto the SPME fiber. This method is rapid yet sensitive and can be completed in 15 min on a 5-mL sample. SPME/GC/MS analysis yielded good reproducibility (RSD > 11%) for each analyte from urine, blood, and milk. Method detection limits for the various biological fluids were determined and ranged from 0.40 ppb for 2,4,6-trimethylaniline in urine to 7.7 ppb for aniline in blood. This SPME sampling protocol can be applied to the biomonitoring of monocyclic aromatic amines from occupational, environmental, and medical exposure.