Auxins are plant hormones that play key roles in almost all growth and developmental processes, such as cell division, elongation, differentiation, and environmental responses. However, biosynthetic pathways and regulatory mechanisms remain unclear. The indole-3-pyruvic acid (IPyA) pathway, from L-tryptophan (Trp) via IPyA, is the main biosynthetic pathway of the natural auxin indole-3-acetic acid (IAA). In this pathway, IAA is biosynthesized from Trp through two enzymatic reactions: aminotransferase (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 [TAA1]/ TRYPTOPHAN AMINOTRANSFERASE-RELATED [TARs]) and YUCCAs (YUCs), which are flavin-containing monooxygenases. We developed the inhibitors of TAA1/TARs and YUC and analyzed the physiological functions of the IPyA pathway using the biosynthesis inhibitors as chemical probes. This paper also describes the regulatory mechanism of the two-step enzymatic reactions in auxin biosynthesis, employing the novel IPyA analog compounds.