The limits of quantitation of the assay of tobramycin in serum by the fluorescence polarization immunoassay system marketed by Abbott Laboratories (TDxFLx system) are 0.1 and 10.0 mg/L. For some pharmacokinetic studies, however, a more sensitive analysis is needed. The sensitivity of the TDxFLx system can theoretically be increased 10-fold by pipetting buffer solution into the sample well and 450 microliters serum into the predilution well. The assay modified in this way can be run with the usual calibration of the apparatus for normal analysis. The analytical performance of this modification of the TDxFLx assay was assessed. Tobramycin concentrations ranged from 0.01 to 1.0 mg/L. Analytical recovery ranged from 85 to 95%. The coefficients of variation for within-run and between-run precision ranged from 0.5% to 5% and from 2% to 6%, respectively, for control concentrations ranging from 0.05 to 0.75 mg/L. Based on recovery and precision results, the lower limit of quantitation was established as 0.025 mg/L. There was no significant detectable cross-reactivity from ceftazidime, aztreonam, flucloxacillin, cilastatin, or imipenem. There was a small, but significant cross-reactivity from gentamicin and netilmicin. Hyperbilirubinemia did not affect the assay, but hyperlipidemia gave falsely elevated results of the tobramycin assay. It was concluded that modification of the assay resulted in an acceptable method to quantify low concentrations of tobramycin in serum.