We develop heterodyned dispersed vibrational echo spectroscopy (HDVE) and demonstrate the new capabilities in biophysical applications. HDVE is a robust ultrafast technique that provides a characterization of the real and imaginary components of third-order nonlinear signals with high sensitivity and single-laser-shot capability and can be used to extract dispersed pump-probe and dispersed vibrational echo spectra. Four methods for acquiring HDVE phase and amplitude spectra were compared: Fourier transform spectral interferometry, a new phase modulation spectral interferometry technique, and combination schemes. These extraction techniques were demonstrated in the context of protein amide 1 spectroscopy. Experimental HDVE and heterodyned free induction decay amide 1 spectra were explicitly compared to conventional dispersed pump-probe, dispersed vibrational echo, and absorption spectra. The new capabilities of HDVE were demonstrated by acquiring single-shot spectra and Melting Curves of ubiquitin and concentration-dependent spectra of insulin suitable for extracting the binding constant for dimerization. The introduced techniques will prove particularly useful in transient experiments, studying irreversible reactions, and micromolar concentration Studies of small proteins.