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A high-performance liquid chromatographic method for the determination of chloramphenicol using an alkaline salt-induced phase separation extraction is described. The extraction procedure is as simple as miscible organic solvent deproteinization methods, yet produces extracts that have negligible protein and minimal interference from acidic compounds. The use of 2,4-dinitroacetanilide as an internal standard and of microprocessor-stored standard relative response factors results in a more precise assay without need for daily routine standardization. Serum is mixed with an equal volume of acetonitrile containing the internal standard, and then solid sodium chloride containing 20% of sodium phosphate by weight is added and mixed. The resulting upper phase is chromatographed on a C-18 reversed-phase column and monitored at 278 nm. Between-day precision is excellent. Comparison with an alternate method and with drug-monitoring proficiency samples showed the method to have excellent accuracy. The possible use of this phase separation extraction in other assays in which miscible solvent extraction is used is discussed.