Abstract: Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is a catalytic enzyme that involves synthesis of collagen and extracellular matrix interactions. Aberrant expression of P4HA1 promotes carcinogenetic invasion and metastasis in breast cancer. In this study, we combined the transcriptomic and drug repositioning approach to intervention new targeted anti-cancer therapy for breast cancer. The mRNA expression, copy number variation, mutation, and clinical patient’s outcome of P4HA1 validated through cBioportal. High-throughput virtual screening and MM-GBSA analysis were performed with Drugbank approved molecules (9,612) for identifying the potent therapeutic drug molecules against P4HA1 using Schrodinger. The cBioportal based gene expression of P4HA1 in the TCGA-breast cancer cohort revealed significant elevated expression in the breast tumor compared to the normal. Subsequently, the high copy number amplification and mRNA expression were high in the invasive breast carcinoma than the other subtypes. In addition, the overall survival was validated with median P4HA1 expression and conferred with poor prognosis of breast cancer patients. Further, receptor-based virtual screening identified top hits of aminoglycoside derivatives, amikacin (glide score −9.58 kcal/mol) and gentamicin (glide score −7.02 kcal/mol) with best docking score and stable interaction with favorable amino acid residues of P4HA1 includes Glu171, Asp178, Lys213, Lys 206, and Leu174. Moreover, both the drug passed the drug-likeness property (ADME) and MM-GBSA energy model. This study integrates genomic and molecular docking approach, suggesting P4HA1 as a prognostic biomarker and selective inhibition might be therapeutically involved in the breast cancer intervention.