A comparative study of the performance of reversed-phase liquid chromatography with micellar-organic (MLC) and aqueous-organic (RPLC) mobile phases is reported for the separation of 16 β-blockers (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol, and timolol). MLC with hybrid mobile phases of sodium dodecyl sulfate (SDS) and propanol is revealed as a very competitive technique for the screening of these drugs. Using a conventional Spherisorb C18 column, the theoretical plates ( N) and asymmetry factors ( B/ A) for the optimal mobile phase compositions were in the ranges N=2200–4400 and B/ A=1.0–1.3 for SDS–propanol against N=290–960 and B/ A=2.2–3.6, and N=1700–5100 and B/ A=1.5–2.2 for acetonitrile–water in the absence and presence of 0.1% (v/v) triethylamine. With a base-deactivated XTerra MS C18 column and acetonitrile–water, peak shape was improved ( N=1100–8800 and B/ A=1.1–2.5), but the elution pattern (large differences in retention between polar and low polar compounds) was similar to that achieved with the Spherisorb column. A set of 14 β-blockers showing a wide range of polarities ( log P o/w =− 0.03 for atenolol to 2.60 for propranolol) could be resolved isocratically with a mobile phase of 0.10 M SDS–15% (v/v) propanol with an analysis time of 32 min. The resolution was better than with aqueous-organic RPLC using the conventional and base-deactivated C18 columns, where only 10–12 compounds could be separated in a single chromatogram. Using specific mobile phases of SDS–propanol, all possible binary mixtures of the 16 β-blockers could be resolved fully (peak purity of the peak pairs, R>0.999), except practolol–sotalol ( R=0.992), nadolol–pindolol ( R=0.959), and esmolol–metoprolol ( R=0.956), which showed a small overlapping. The resolution of the individual peaks with the aqueous-organic mixtures was more problematic.