Our understanding of the relationship between cancer initiation and subsequent progression to malignancy remains elusive. Biological signaling pathways are extremely complex with a multitude coding and non-coding oligonucleotides and proteins working in concert. Highly sensitive and selective spectroscopic instrumentation and probes are needed to resolve the various biomarkers contributing to the signaling processes. Multiphoton spectroscopy benefits from reduced scattering of light in the red region of the spectrum and excellent analytical signal to background levels. The excellent sensitivity associated with multiphoton spectroscopy makes it an attractive candidate for biological and tissue imaging applications. Analytical merits of nanostar contrast agents, ophthalmic compounds, and genetic biomarker probes will be discussed. Nanomaterials have demonstrated improved contrast properties compared to traditional fluorophores. The emission characteristics, limits of detection, and proof of principle for imaging applications of nanostars will be addressed. Nanostars demonstrated transitions from non-linear to linear absorption processes as the power was increased. Photoluminescence spectra as a function of excitation power and emission wavelength were examined for their contribution to the observed multitude of nonlinear processes. Sometimes adding in additional probes can interfere with normal biological function or be harmful to the patient in medical applications. To address such concerns label free spectrochemical analysis techniques are in demand for pharmaceutical and other biomedical applications. The potential and feasibility of multiphoton label free analytical measurement and imaging of ophthalmic compounds in ocular tissue will be discussed. Rapid methods to detect gene expression in complex samples are in high demand. An atypical Förster Resonance Energy Transfer (FRET) pair combination as labels for DNA probes will be presented. The probes were designed to bind the ERBB-2 gene, a biomarker for breast cancer. Hybridization of the FRET pair probes to the ERBB-2 target sequence resulted in the FRET pairs being adjacent to each other leading to a reduction of the fluorescent signal. The probe combination demonstrated sensitive and specific recognition of ERBB-2 genes within two minutes.