Copyright © 2016 Caleb R. Dulaney et al. Tremendous technological advancements in prostate radiotherapy have decreased treatment toxicity and improved clinical outcomes for men with prostate cancer. While these advances have allowed for significant treatment volume reduction and whole-organ dose escalation, further improvement in prostate radiotherapy has been limited by classic techniques for diagnosis and risk stratification. Developments in prostate imaging, image-guided targeted biopsy, next-generation gene expression profiling, and targeted molecular therapies now provide information to stratify patients and select treatments based on tumor biology. Image-guided targeted biopsy improves detection of clinically significant cases of prostate cancer and provides important information about the biological behavior of intraprostatic lesions which can further guide treatment decisions. We review the evolution of prostate magnetic resonance imaging (MRI) and MRI-ultrasound fusion-guided prostate biopsy. Recent advancements in radiation therapy including dose escalation, moderate and extreme hypofractionation, partial prostate radiation therapy, and finally dose escalation by simultaneous integrated boost are discussed. We also review next-generation sequencing and discuss developments in targeted molecular therapies. Last, we review ongoing clinical trials and future treatment paradigms that integrate targeted biopsy, molecular profiling and therapy, and prostate radiotherapy.