Background: Wound age determination is a critical aspect of forensic investigations. The primary objective aimed to determine the appropriateness of VEGF, α-SMA, TNF-α, and TGF-β1 biomarkers for wound age determination in rats. We conducted histopathological, immunohistochemical, and molecular assessments to investigate changes in these markers over different post-wounding time intervals. Methods: Forty-eight healthy adult male albino rats were separated into 8 equal groups based on the post-wounding time interval. Histopathological examinations, immunohistochemical staining, and gene expression analyses were performed on skin samples to assess the statement of transforming growth factor beta (TGF-β1), tumour necrosis factor-alpha (TNF-α), alpha-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF). Data were analysed statistically to determine the significance of changes over time. Results: The histopathological analysis revealed distinct changes in skin tissue over time, ranging from ulceration to reepithelialization and tissue regeneration. Immunohistochemical examination demonstrated varying levels of VEGF and α-SMA expression, with marked increases in later post-wounding periods. Molecular analysis indicated a significant upregulation of TNF-α and TGF-β1 expression, reaching peak levels around seven days post-wounding. Conclusions: The use of VEGF, α-SMA, TNF-α, and TGF-β1 as potential biomarkers for wound age determination. The expression patterns of these markers in the rat skin suggest their suitability for forensic applications.