Acanthamoeba are ubiquitous protists that play an environmental role in regulating microbial diversity; they also occasionally cause infections of the eye (Acanthamoeba keratitis) and brain (granulomatous amoebic encephalitis). These organisms exhibit two distinct phenotypes. The trophozoite form dominates in favorable conditions, in which the Acanthamoeba move through the extension of pseudopodia, engulfing microbes and other particles. During stressful conditions, the Acanthamoeba undergo a process of encystment, in which they build a double cell wall and become relatively inactive. The cyst form can survive years until more favorable conditions arise, at which point they may excyst. For this study, multiple laboratory encystment methods were compared to determine the percent encystment and the different viabilities of laboratory-produced cysts. Furthermore, four different encystment genes were targeted for development of a primer library for reverse-transcription, polymerase chain reaction expression studies. The library was developed using sequences accessed from various databases, including NCBI and EMBL; primers were screened through polymerase chain reaction, and those primers producing positive results were used to further screen cellular RNA that was extracted from encysting cells over various time points during the encystment process, and using various encystment media. Using these methods, target gene involvement in the encystment process was compared between species and encystment methods. These studies lay the foundation for quantitative gene expression analysis, and provide the basis for comparison of various encystment methods.