Two unanswered questions in the field of Chlamydia are 1) How does the inclusion grow over time? and 2) What promotes RB-to-EB conversion? Small molecule compounds have been widely used to manipulate infection conditions to determine specific targets required for these stages of chlamydial development. Although there are many publications describing the effects of anti-chlamydial inhibitors, the lack of a standard approach makes it challenging to compare these effects across studies.In chapter 3, our manuscript (submitted) defines the weaknesses in the current methodologies that the Chlamydia field implements to study inhibitor treatments during the infection. We additionally demonstrate approaches to yield more information from inhibitor studies to more accurately describe the ways different conditions alter the infection. Using published and novel chlamydial inhibitors, we found that analysis with one assay at one or two time points in the infection is insufficient because inhibitors can change the length of the infection. Furthermore, we showed that phenotypes can vary depending on the time of analysis and the assay used. To circumvent these issues, we propose a novel measurement, Progenymax, which takes changes in the length of the developmental cycle into consideration and can easily be used to compare differences in progeny, regardless of the condition. In chapter 4, we describe a novel inhibitor of the chlamydial infection, H89, which slowed inclusion growth and caused a block in RB-to-EB conversion. We tested several known kinases and pathways that were previously reported to be blocked by H89, but none were inhibited by our H89 treatment conditions. In chapter 5, we continued to explore the mechanisms targeted by H89 to elucidate why the inhibitor was such a strong anti-chlamydial inhibitor. We found that H89 causes lysosomal accumulation of cholesterol, which correlated with a defect in lysosomal acidification. These studies suggest that H89 prevents lysosomal acidification, resulting in cholesterol accumulation. Previous studies suggest that cholesterol may be necessary for the chlamydial infection, meaning these H89-mediated defects on inclusion growth and progeny production are likely due to a decrease in cholesterol transport out of the lysosome and reduced cholesterol accessibility by chlamydiae.