Hyperspectral Imaging (HSI) is a technique that can be used with darkfield microscopy for investigating biological interactions of optically active nanoparticles (NP) based on their spectral signatures. The objective of this study is to investigate the capabilities of HSI technology to characterize (1) spectral characteristics of gold, silver, and manganese NPs, (2) stability of gold NPs in exposure media and growth media based on spectral shifts, and (3) cellular interaction of NPs by correlating spectral data between NPs in water or media and NPs exposed to cells. Results demonstrate the unique spectral characteristics of the NPs investigated. Gold and silver NPs both display plasmonic properties; however, gold NPs exhibited a more narrow spectral profile. Silver NPs demonstrated several distinct scattering spectra due to the strong dependence on morphology. Manganese NPs do not display plasmonic properties, but still exhibited a unique spectral response, allowing them to be detected in cells. Due to the narrow spectral profile for gold NPs, stability was investigated in biological media, demonstrating the ability for HSI to detect the stabilizing effect of serum on NPs. Cellular interaction studies showed that the peak scattering wavelength for NPs in cells deviated from NPs in water or media, but NPs were still able to be identified based on intensity and shape of the scattering curves. Overall, HSI was demonstrated as a useful technique for evaluating optically active NPs in biological media and cellular environments.