The testis is a complex organ responsible for sperm production and hormone synthesis. Spermatogenesis, the process of sperm formation, occurs in the seminiferous tubules and involves the coordinated development of germ cells with the support of Sertoli cells. These provide structural and nutritional support for germ cells, regulate spermatogenesis, and maintain the blood-testis barrier. Despite extensive research on Sertoli cells, their protein expression patterns and spatial localization in relation to other testicular cell types remain poorly characterized. This project aimed to investigate the expression patterns and spatial localization of proteins in Sertoli cells. Single-cell RNA sequencing data and immunohistochemistry stainings from the Human Protein Atlas were used to choose candidate proteins to be visualized in a multiplex Immunofluorescence panel. After staining, the slides were scanned and analyzed manually as well as through automated image analysis via QuPath. 101 proteins were successfully stained and localized within testis tissue and subcellular locations in Sertoli cells were annotated. Additionally, a list of uncharacterized proteins in Sertoli cells were identified. The employed workflow demonstrated its potential for identifying Sertoli cell-specific proteins and can be applied to future research. Additional validation is required to verify the presence and identify function of these proteins in Sertoli cells. For this purpose, techniques such as mass spectrometry-based proteomics and mouse knock-out models could be used.