Novel nonlinear multi-photon laser spectroscopic methods are presented for ultrasensitive detection, separation and identification of biomarker proteins for early diagnosis of multiple sclerosis (MS), cancer, metabolites of carcinogenic molecules, and nicotine and their metabolites. Using compact, portable capillary- or microchip-based separation methods, we demonstrate excellent detection sensitivity and chemical specificity levels. Laser wave mixing is an unusually sensitive optical absorption-based detection method that offers significant inherent advantages including excellent sensitivity, small sample requirements, short optical path lengths, high spatial reso lution, and high spectral resolution. Based on the quadratic dependence of the signal on analyte concentration, it allows more dramatic monitoring of small changes in analyte properties. Wave mixing detection sensitivity is comparable or better than thos e of fluorescence-based methods and enzyme-linked immunoassays for clinical diagnostic and screening. Since the wave-mixing signal is a laserlike coherent beam, it can be collected with virtually 100% efficiency and high signal-to-noise ratio. Laser excitation wavelengths can be tuned to detect multiple analytes in their native forms. Our analysis time is short (minutes) as compared to those of other conventional methods of separation, e.g., sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). When wave mixing is coupled to a high-resolution capillary- or microchip-based separation system, disease biomarkers can be separated and identified at the zepto-mole levels. Laser wave mixing allows for sensitive detection of cancer biomarkers through complexation of cancer antigen with chromophores and absorptionmodifying tags.