A major advantage of flow cytometry is its flexible and open instrument configuration, which is highly suitable for systems integration. This flexibility permits the coupling of auxiliary instrumentation that may offer the measurement of parameters other than those typically measured by this multiparameter measurement technique. On the basis of this advantage, we explore the principle and application of hyperspectral imaging (HSI), which has the potential to be a useful add-on feature to flow cytometry applications. Application of HSI to flow cytometry involves the acquisition of spatial information and rendering it in spectral form. In this work, we describe the development and application of an HSI system which provides both spectral and spatial information. Spectral information was generated by obtaining an entire spectrum of a single sample site within a wavelength region of interest, while spatial information was generated by recording a two-dimensional (2D) image of an area of the sample of interest at one specific wavelength. HSI is a promising additional feature to flow cytometry since it can provide both spatial (image format) and spectral information in addition to the multiparameter information already available from flow cytometry measurements.