The rising incidence of prostate cancer necessitates innovative treatment approaches, particularly as diseases like the COVID-19 pandemic can disrupt traditional cancer care. This study aimed to evaluate the impact of hypofractionated versus conventionally fractionated radiotherapy on prostate cancer cell lines in vitro. Prostate cancer cell lines (PC-3 and DU-145) were exposed to varying doses of radiation alongside non-cancerous BPH-1 cells. We assessed radiation effects on cell proliferation, viability, colony formation, DNA repair, migration, invasion, and cytotoxicity. Results demonstrated that the prostate cell lines exhibited varying responses, with hypofractionation favourably impacting aggressive PC-3 cells while preserving non-cancerous cells. In contrast, conventional fractionation led to increased invasion and cytotoxicity in both prostate cancer cell lines. These findings advocate for personalised radiation therapy approaches that enhance treatment efficacy by considering the distinct behaviours of differing prostate cancer subtypes.