A single gradient LC method for the determination of related substances in both saquinavir (SQV), saquinavir mesilate (SQVM) has been published in a consultation document of the International Pharmacopoeia, WHO Drug Information. The method uses a base deactivated reversed phase C18 column (25 cm × 4.6 mm i.d.), 5 μm kept at a temperature of 30 °C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 220 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards SQV(M) components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm × 4.6 mm i.d.), 5 μm. A central composite design was applied to examine the robustness of the method. The method shows good precision, linearity, sensitivity and robustness. SQV(M) commercial samples of bulk drugs were examined using this method.