The regulation of auxin (indole-3-acetic acid, IAA) level in plants is a complex mechanism and the concept is not completely understood. There are many lines of evidence suggesting that amino acid conjugates of IAA play a vital role in auxin homeostasis, but the involved plant enzymes are still under investigation. In the present study, the alteration in the levels of auxin and its conjugates in the different physiological stages and organs, as well as its role in flower bud abscission in Pistacia vera L., were investigated. The levels of the auxin and its conjugates, such as IAA, indole butyric acid (IBA), IAA-Ala, IAA-Leu, IAA-Asp, and IAA-Glu, were analyzed by ultraperformance liquid chromatography-electrospray ionization-mass spectrometry (UPLC/ESI-MS/MS) technique. Among the auxin conjugates, IAA was the main conjugate and dominant indole detected in pistachio extract observed in this work. It has also been determined that quite a few organs of 'Off-year' trees have higher auxin content than 'On-year' trees. It was observed that the auxin level is low in fruit-bearing trees in both leaves and shoots. It affects fruit yield and fruit bud abscission and plays some role in the alternate bearing. Decreased levels of auxin were observed in most of the organs of 'On-year' trees during kernel development resulting in bud abscission. This study shows that auxin conjugates play an important role in IAA metabolism, particularly in flower bud abscission and fruit kernel development.