Auxin is the essential plant hormone that regulates many aspects of plant growth and development. Plants typically possess highly complex biochemical networks to regulate the homeostasis of the active hormone, through the regulation of biosynthesis, degradation, transport and conjugation. Biosynthesis, among other processes, has been of particular importance and warranted extensive studies over the decades of auxin research. A number of pathways were proposed and some enzymes potentially involved have been characterized. Stable isotope labeling and turnover studies have proven very useful in these investigative efforts. With the advancement of analytical and computational technologies, it is now feasible to concurrently analyze the turnover patterns of all the precursors in the entire auxin biosynthesis network. I devoted chapter two of the dissertation to establish LC-MS methods to concurrently quantify most of the auxin precursors and deployed different isotopic labeling strategies for turnover studies in <italic>Arabidopsis thaliana<italic>. Preliminary results showed that indole-3-pyruvate (IPyA) and indole-3-acetaldehyde (IAAld) were among the fastest to be labeled and a key regulatory step existed between IPyA and indole-3-acetic acid (IAA). Chapter three reported the discrepancy and the study of the amount of the total IAA determined by alkaline hydrolysis and that of the summation of all known forms of IAA conjugates in <italic>Arabidopsis<italic>. My results indicated that chemical artifacts induced by harsh chemical treatments were responsible for a significant portion of the unknown putative IAA conjugates. In chapter four, I described a facile way to directly survey a plant extract for its indole profile, notably IAA conjugates, based on high resolution and accurate mass (HR/AM) liquid chromatography-mass spectrometry (LC-MS). The method was successfully applied to <italic>Glycine max<italic>, <italic>Solanum lycopersicum<italic>, <italic>Cocos nucifera<italic>, and <italic>Ginkgo biloba<italic>. Together, these investigations and developments have led to an improved understanding of auxin metabolism and now provide useful tools for subsequent studies.