Gold nanoparticles (GNPs) have been stabilized with nonionic surfactant ligands, i.e., Brij 35, and their aggregation could be induced rapidly and selectively by biologically active low-molecular-mass thiols including sulphydryl-containing amino acids (cysteine and homocysteine) and small peptides (glutathione, cysteinylglycine, and glutamylcysteine). A new postcolumn detection method has been developed for high-performance liquid chromatography (HPLC) assay of these small biothiols based on the analyte-induced aggregation of the GNPs. Compared with conventional thiol-reactive probes, the GNP colloids are easier to prepare, much more stable in aqueous solution over a wide pH range and at ambient temperature, and exhibit relatively high selectivity toward small biothiols. The analysis of human urine samples demonstrated that the proposed method is promising in HPLC assay of the small thiol molecules in biological fluids.