The amide I Raman tensor corresponding to the antiparallel β-sheet structure in proteins has been determined by polarized Raman microspectroscopy of fowl feather rachis using polarized Raman spectra excited in the near-infrared (785 nm). For a Raman tensor principal axis system (XYZ), in which X is perpendicular to the plane of the pleated β-sheet, Y is in the plane of the sheet and perpendicular to the direction of the polypeptide chain, and Z is parallel to the direction of the polypeptide chain, the principal tensor components (αXX, αYY, αZZ) are found to satisfy the following relationships: R1 ≡ αXX/αZZ = 0.32 and R2 ≡ αYY/αZZ = 3.48. With this Raman tensor determination, we show that semiquantitative estimates of the total antiparallel β-sheet content in β-rich proteins can be extracted from polarized Raman intensity measurements on the amide I marker of the β-sheet occurring near 1664 cm−1. We demonstrate this approach for the β-rich silk proteins of the silkworm and spider. Copyright © 2006 John Wiley & Sons, Ltd.