Localized production of signaling agents is an essential feature for living cells. Many of these signaling molecules are lipid entities. However, due to their hydrophobic nature and highly diverse cellular functions, certain potent, low-abundance lipids that act as signaling agents remain understudied with traditional biochemical techniques. Thus, the advancement of modern chemical biology tools represents a promising perspective to tackle these complex biological problems. Chapter 1 summarizes the challenges and recent advancement to study phospholipid signaling and discusses its relevance towards cell signaling and lipid trafficking. In Chapter 2, the author discusses the design, synthesis and characterization of a novel, bioorthogonal chemistry-based strategy, termed RT-IMPACT, to image the biosynthesis of a specific signaling lipid called phosphatidic acid (PA). With fast chemical kinetics and optimal enzyme specificity, PA produced by phospholipase D (PLD) enzyme activity can be visualized in living cells in real-time. The author further demonstrates that RT-IMPACT is capable of accurately reporting subcellular locations of PA production in response to different upstream stimuli. In Chapter 3, the author applies RT-IMPACT tools to investigate PTHR signaling that are previously underexplored. The author shows that PLD activation is specifically downstream of PTHR-Gq protein signaling. Moreover, the Gq signaling pathway, in stark contrast to the Gs pathway, is transient and localized on the plasma membrane as revealed by time-resolved imaging of PLD activities. Lastly, through inhibition of endocytic pathways, a competitive relationship between the Gq and Gs pathway is revealed. In Chapter 4, the author characterizes a family of lipid transfer proteins—ESyts—that are responsible for trafficking of unnatural fluorescent lipids from PM to ER. A positive correlation between the expression levels of ESyts and trafficking rates from PM to ER is established. Next, through protein engineering, lipid transfer activities of ESyts are shown to be directly responsible for removal of unnatural fluorescent lipids from the PM. In the final chapter, the author summarizes the major findings and significance of this work towards understanding of physiologically relevant pathways. Several potential future directions, including screening for potential upstream stimuli and identifying other lipid transfer proteins, are briefly described.