The origins of epithelial ovarian cancer (EOC) have long been debated, with proposed sources including ovarian surface epithelial (OSE) cells, secondary Müllerian tract structures, or fallopian tube epithelium. Despite being the second most common gynecological cancer and a leading cause of death in the United States, in vitro cell models mimicking normal ovarian epithelial cells and their malignant counterparts are lacking. To address this gap, we established immortalized human OSE (IHOSE) cell lines that demonstrate stable in vitro growth without malignant properties. IHOSE cell lines were unique cell lines by analyzing short tandem repeat (STR) profiling. In addition, the epithelial characteristics were confirmed by cytokeratin 7 and cytokeratin 18 marker expression. IHOSE cell lines were subjected to Opal multiplex immunohistochemistry (IHC) analysis, which established four distinct subtypes based on marker dominance. These studies offer the most basic but essential cellular characterization information for IHOSE cell lines, providing critical data that can guide the selection of cells when inducing normal controls or disease models.