The near-infrared (NIR) spectral region (700-2500 nm) is a fertile source of chemical information in the form of overtone and combination bands of the fundamental infrared absorptions and low-energy electronic transitions. This region was initially perceived as being too complex for interpretation and consequently was poorly utilized. Advances in chemometric techniques that can extract massive amounts of chemical information from the highly overlapped, complex spectra have led to extensive use of NIR spectrophotometry (NIRS) in the food, agriculture, pharmaceutical, chemical, and polymer industries. The application of NIRS in clinical laboratory measurements is still in its infancy. NIRS is a simple, quick, nondestructive technique capable of providing clinically relevant analyses of biological samples with precision and accuracy comparable with the method used to derive the NIRS models. Analyses can be performed with little or no sample preparation and no reagents. The success of NIRS in any particular case is determined by the complexity of the sample matrix, relative NIR absorptivities of the constituents, and the wavelengths and regression technique chosen. We describe the general approach to data acquisition, calibration, and analysis, using serum proteins, triglycerides, and glucose as examples.