A unique probe designed to acquire nuclear magnetic resonance difference spectra of two samples is presented. The NMR Difference Probe contains two sample coils in a resonant circuit that switches between parallel excitation and serial acquisition to cancel common signals such as solvent peaks and impurities. Two samples containing a common analyte, acetonitrile, were used to demonstrate signal cancellation in a difference spectrum collected with a single pulse experiment. The cancellation was over 96% effective. The approach described has applications in the areas of solvent subtraction and spectral simplification.