A sensitive plasma assay for acyclovir has been developed and validated. Acyclovir was separated from plasma components using Oasis HLB columns. Separation was obtained with no plasma interference using micellar electrokinetic chromatography (175 mM SDS) and hydroxypropyl-beta-cyclodextrin (100 mM) in 90 mM borate buffer (pH 8.8) containing 0.2% NaCl. High sensitivity was achieved by large volume sample introduction and stacking. The linear range was from 20 to 10000 ng/ml with a limit of quantitation of 20 ng/ml. This method is a viable alternative to HPLC because of its high separation and sensitivity, reproducibility, and adaptability to other nucleoside analogs.