A liquid chromatographic method is described for analysis of benzocaine (BZ), a proposed fish anesthetic, in rainbow trout plasma. Mean recoveries of BZ from plasma samples fortified at 44–10 100 ng/mL were 96–100%. The method detection limit is 10 ng/mL, and the limit of quantitation is 37 ng/mL. Acetylation of BZ occurs in whole blood after storage at room temperature (i.e., 21 °C) for 10 min. However, no acetylation of BZ was detected in plasma samples held at room temperature for 4 h. Mean method precision for plasma samples with incurred BZ residue is similar to that for fortified samples in the same concentration range (relative standard deviations of 0.9 and 1.2%, respectively).