The HPLC–MS/MS method was developed to profile the dynamics of abscisic acid (ABA) and ABA-glucose ester (ABA-GE) after cloning glycosyltransferase enzyme family gene AtUGT71C5 into Arabidopsis thaliana. By constructing over-expression lines (OE) and down-expression lines (DN), we acquired mutant strains to analyze the function of AtUGT71C5. The multiple-reaction monitoring (MRM) was used for quantitative determination in negative mode. The transition was m/z 263.1→153.0 for ABA ([M–H]+), m/z 425.1→263.0 for ABA-GE ([M–H]+), and m/z 321.0→152.0 for chloramphenicol. The linear range was 0.8684–217.1 ng/mL for ABA and 0.3920–196.0 ng/mL for ABA-GE. The accuracy was 88.0–109.0% for ABA and 86.6–113.0% for ABA-GE; the inter-day and intra-day precisions were less than 5.4% for ABA and 8.9% for ABA-GE, respectively. This method is simple and sensitive enough for determination of ABA and ABA-GE in A. thaliana leaves. All the evidence confirmed the speculation that AtUGT71C5 can mediate abscisic acid homeostasis. Keywords: HPLC–MS/MS, ABA, ABA-GE, AtUGT71C5