Diffuse-reflectance Fourier-transform mid-infrared spectroscopy (MidIR) can identify the presence of important organic functional groups in soil organic matter (SOM); however, spectral interpretation needs to be validated to correctly assess changes in SOM quality and quantity. We amended soils with known standards, increasing the total C in the sample by 50%, and measured changes in MidIR spectra. Adenine, casein, cellulose, ergosterol, glucosamine, glycine, guanine, indole, methionine, palmitic acid, egg protein, chlorophyllin, tannic acid, xylose, urease, and vanillin standards were used. In addition, corn (Zea mays L.) stalk feedstock and two chars produced at different temperatures were studied. Two soils were used: a Hoytville, OH, soil (2.5% C and 36% clay) and an Akron, CO, soil (1.5% C and 14% clay). The addition of standards with >10% N content resulted in increased amide-like absorbance at 1670, 1588, and 1513 cm−1. Bands at 2970 to 2800, 2200 to 2000, and 1030 to 1160 cm−1 were sensitive to added polysaccharide. Protein addition increased absorption at 2970 to 2800 cm−1 but also increased the 1691 and 1547 cm−1 amide bands. Vanillin addition resulted in higher absorbance at the 1592, 1515, and 1295 cm−1 aromatic C=C bands. Biochars produced at 300°C resulted in increased absorbance at carbonyl and aliphatic bands, while addition of 500°C biochar increased aromatic absorbance. Our results showed that MidIR is sensitive to relatively small changes in SOM. If assumptions about the soil mineralogy are met, specific spectral bands can be used to follow changes in SOM chemistry.