Parasites change their transcriptional systems in different developmental stages and in response to environmental changes. To investigate the molecular mechanisms that underlie transcriptional regulation, it is essential to identify the exact positions of the transcriptional start sites (TSSs) and characterize the upstream promoter regions. However, it has been essentially impossible to obtain comprehensive information using conventional methods. Here, we introduce our TSS-seq method, which combines full-length technology, oligo-capping, and rapidly developing next-generation sequencing technology. TSS-seq has enabled identification of TSS positions and upstream promoter activities as digital TSS tag counts within a reasonable cost and time frame. In this chapter, we describe in detail the TSS-seq method for the identification and characterization of the promoters in Toxoplasma gondii.