For measurement of tylosin, ammonia nitrogen, and glucose concentration during the culture of Streptomyces fradiae using Near Infrared Spectroscopy, the calibration using various mathematical models was performed and then, based on the linear model, the validation was carried out. In the case of sucrose concentration using the MLR method, the Standard Error of Prediction and Multiple correlation coefficient were 1.97, and 0.991, respectively. In the case of ammonia nitrogen concentration using the PLSR method, the Standard Error of Prediction and Multiple correlation coefficient were 0.13, and 0.990, respectively. In the case of tylosin concentration using the PLSR method, the standard Error of Prediction and Multiple correlation coefficient were 0.54, and 0.984, respectively.