An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC-MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g(-1) fresh weight (IAA) to 0.44 pmol g(-1) fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.