Multi-photon degenerate four-wave mixing is demonstrated as an ultrasensitive absorption-based optical method for detection, separation and identification of biomarker proteins in the development of early diagnostic methods for HIV- 1, cancer and neurodegenerative diseases using compact, portable microarrays and capillary- or microchip-based chemical separation systems that offer high chemical specificity levels. The wave-mixing signal has a quadratic dependence on concentration, and hence, it allows more reliable monitoring of smaller changes in analyte properties. Our wave-mixing detection sensitivity is comparable or better than those of current methods including enzyme-linked immunoassay for clinical diagnostic and screening. Detection sensitivity is excellent since the wave-mixing signal is a coherent laser-like beam that can be collected with virtually 100% collection efficiency with high S/N. Our analysis time is short (1-15 minutes) for molecular weight-based protein separation as compared to that of a conventional separation technique, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When ultrasensitive wavemixing detection is paired with high-resolution capillary- or microchip-based separation systems, biomarkers can be separated and identified at the zepto- and yocto-mole levels for a wide range of analytes. Specific analytes can be captured in a microchannel through the use of antibody-antigen interactions that provide better chemical specificity as compared to size-based separation alone. The technique can also be combined with immune-precipitation and a multichannel capillary array for high-throughput analysis of more complex protein samples. Wave mixing allows the use of chromophores and absorption-modifying tags, in addition to conventional fluorophores, for online detection of immunecomplexes related to cancer.