The current clinical standard for mass screening of prostate cancer are prostate-specific antigen (PSA) biomarker assays. Unfortunately, the low specificity of PSA's bioassays to prostate cancer leads to high false-positive rates, as such there is an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we have successfully demonstrated, with the use of our Photonic-Crystal based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, detection of prostate cancer (PC-3) cells against benign prostate hyperplasia (BPH-1) cells. The PC-TIR biosensor achieved detection of individual prostate cancer cells utilizing cellular refractive index (RI) as the only contrast parameter. To further study this methodology in vitro, we report a comprehensive study of the cellular RI's of various prostate cancer and noncancerous cell lines (i.e. RWPE-1, BPH-1, PC-3, DU-145, and LNCaP) via reflectance spectroscopy and single-cell RI imaging utilizing the PC-TIR biosensor. Our study shows promising clinical potential in utilizing the PC-TIR biosensor system for the detection of prostate cancer against noncancerous prostate epithelial cells.