The invention provides methods for calibrating spectral data acquisition systems. These calibration methods produce spectral data sufficiently accurate for use in tissue classification algorithms. The invention improves the accuracy of spectral-based tissue classification schemes, in part, by properly accounting for spatial variations, instrument-to-instrument variations, and patient-to-patient variations in the acquisition of spectral data from tissue samples. Effects that are accounted for include, for example, stray light effects, electronic background effects, variation in light energy delivered to a tissue sample, spatial heterogeneities of the illumination source, chromatic aberrations of the scanning optics, variation in wavelength response of the collection optics, and efficiency of the collection optics.