Exogenous exposure to estrogenic compounds is implicated in various health conditions, including reproductive disorders, hormone-related cancers, and metabolic disturbances. In healthy males, activation of the estrogen receptors (ERs) can induce feminization and toxicity. However, screening for estrogenic compounds is limited to uterotrophic assays without an established male analog. Studies have reported that exposure to estrogen significantly enlarged the seminal vesicles (SV). However, screening strictly by the wet weight of the SV could obscure the identification of weakly estrogenic or ER-selective compounds. Therefore, protein-based markers are beneficial to complement the weight-based assessments. In this study, we report a discovery-driven proteomic analysis of 17β-estradiol's (E2) effects on the SV for the first time to identify potential biomarkers for screening estrogenic compounds in males. In our study, orchiectomized mice were subcutaneously injected with E2 or the vehicle daily for five days. Proteins from the SV were extracted and analyzed by shotgun proteomics relying on liquid chromatography-tandem mass spectrometry. Acquired tandem mass spectra were searched against the UniProt Mouse protein database using Proteome Discoverer (Thermo Fisher Scientific) and MaxQuant (Max Planck Institute). Verification of identified proteins and relative quantifications were performed using Scaffold (Proteome Software). Post hoc t-tests were performed to identify differently expressed proteins (DEPs) between groups. DEPs were mapped to protein interaction networks and pathways through Ingenuity Pathway Analysis® (IPA®, Qiagen). Our discovery-driven proteomics identified over 7000 proteins with high confidence from the SV of E2-treated and control mice. In addition, bioinformatics-based analysis using IPA® of the E2-regulated proteins showed enrichment in molecular and cellular functions associated with carbohydrate metabolism, DNA replication, repair, and free radical scavenging. Furthermore, corroborating our treatment, IPA®’s molecule activity predictor tool indicated enhanced upstream activation of the ER-β (ESR2). We identified several preclinical biomarkers participating in androgen receptor activity, ER-mediated transcription, and reproductive system development and function. Targeted proteomics-based validation of these biomarkers is ongoing. Our study aims to provide an in-depth account of the alterations occurring at the protein level in response to E2 exposure for screening and validation of preclinical biomarkers with diagnostic potential.