In situ spectral analysis can be used to understand the targeting and interaction of agents in cellular compartments. A range of novel red excitable fluorescent probes, related to the anthraquinone family of anti-cancer agents, were designed for their DNA affinic properties and their ability to enter and penetrate living cells. We report on the spectral features of these probes, both in solution and bound within intact cells, to identify unique fluorescent signatures that exploit their use in bioassays on optical biochip devices. The probes demonstrated red shifted emission spectra and increased 2 photon lifetime, with minimal fluorescent enhancement, upon binding to DNA. Spectral confocal laser scanning microscopy revealed complex emission profiles representing the bound (nuclear) and unbound (cytoplasmic) fractions of the DNA probes within live interphase, mitotic and apoptotic cells. Analysis of the emission peaks encoded the spectra to provide cell compartment recognition and profiles for cells in different cell states. Sampling the entire emission spectra of these probes for cell locating, even in the presence of unbound molecules, provides good signal-to-noise in biochip devices. Furthermore, by sampling the fluorescence output at specific spectral windows we can obtain high spatial information without imaging. The technological challenge is to integrate these fluorophores and appropriate detection capacity onto an optical biochip platform with microfluidic systems for cell handling.