A calibration reference for a multipass Raman analysis system, wherein a combination excitation and collection beam passes through a focal point F0 within a sample volume multiple times, is provided. The calibration reference includes a body of material having a known spectral response when illuminated by the combination excitation and collection beam. The size or shape of the body is selected or modified to keep the focal point at F0 within the body when the body is positioned within the sample volume for calibration purposes.