The objective of our work was to develop and evaluate the performance of a rapid method for measuring fat, protein, moisture, and salt content of Cheddar cheese using a combination mid-infrared (MIR) transmittance analysis and an in-line conductivity sensor in an MIR milk analyzer. Cheddar cheese was blended with a dissolving solution containing pentasodium triphosphate and disodium metasilicate to achieve a uniform, particle-free dispersion of cheese, which had a fat and protein content similar to milk and could be analyzed using a MIR transmittance milk analyzer. Annatto-colored Cheddar cheese samples (34) from one cheese factory were analyzed using reference chemistry methods for fat (Mojonnier ether extraction), crude protein (Kjeldahl), moisture (oven-drying total solids), and salt (Volhard silver nitrate titration). The same 34 cheese samples were also dissolved using the cheese dissolver solution, and then run through the MIR and used for calibration. The reference testing for fat and crude protein was done on the cheese after dispersion in the dissolver solution. Validation was done using a total of 36 annatto-colored Cheddar cheese samples from 4 cheese factories. The 36 validation cheese samples were also analyzed using near-infrared spectroscopy for fat, moisture, and the coulometric method for salt in each factory where they were produced. The validation cheeses were also tested using the same chemical reference methods that were used for analysis of the calibration samples. Standard error of prediction (SEP) values for moisture and fat on the near-infrared spectroscopy were 0.30 and 0.45, respectively, whereas the MIR produced SEP values of 0.28 and 0.23 for moisture (mean 36.82%) and fat (mean 34.0%), respectively. The MIR also out-performed the coulometric method for salt determination with SEP values of 0.036 and 0.139 at a mean level of salt of 1.8%, respectively. The MIR had an SEP value of 0.19 for estimation at a mean level of 24.0% crude protein, which suggests that MIR could be an easy and effective way for cheese producers to measure protein to determine protein recovery in cheese making.