A comparative study of the separation and identification of nine β-blockers in human plasma is presented on C18 and new generation C28 columns. The reported β-blockers are acebutalol, metoprolol, bopindolol, oxprenolol, bufuralol, bisoprolol, indinolol, carazolol and alprenolol. The mobile phase used was phosphate buffer (20.0 mM, pH 7.0)–acetonitrile (75 : 25, v/v) at 0.5 mL min−1 flow rate. The detection was achieved at 220 nm with a working temperature of 27 ± 1 °C. The values of the retention, separation and resolution factors on the C18 column varied from 2.38–14.68, 1.32–1.51, and 1.49–4.91, respectively. On the other hand, these values were 2.04–11.85, 1.23–1.37 and 1.33–6.03 on the C28 column, respectively. The new generation C28 column was found to be better than C18 due to the highly non-polar nature of the former. Attempts have been made to explain the separation mechanism of these columns. The SPE method was selective, efficient, fast and reproducible. The reported β-blockers were found to interact with plasma proteins. The reported SPE and HPLC methods are selective, efficient, rugged, fast and reproducible.