Abstract A procedure for the quantification of surfactant protein B (SP-B) and surfactant protein C (SP-C) in surfactant preparations is described. After butanol extraction of surfactant, the resulting extract is separated by Sephadex LH-60 HPLC, using an elution fluid which consists of 30% dichloromethane, 65% methanol, and 5% 100 m MHCl. SP-B and SP-C are detected by absorbance at 228 nm and quantified using standard stock solutions. Run length is approximately 70 min. Detection limits are 1 μg SP-B and 4 μg SP-C.