Corn steep liquor (CSL) is an important raw material that has a high nutritional value and serves as a nitrogen source. This study aimed to develop a fast, versatile, cheap, and environmentally safe analytical method of quantifying the total acidity (TA) of CSL as well as its contents of dry matter (DM), total sugars (TS), total reducing sugars (TRS), total free amino acids (TFAA), total nitrogen (TN), and total sulfite (TSu). The near-infrared (NIR) spectroscopy measurements of 66 samples (22 batches) of CSL were analyzed by partial least-squares regression using several spectral preprocessing methods. Multivariate models developed in the NIR area showed good predictive abilities for DM, TA, TS, TRS, TFAA, TN, and TSu determination. These results confirm the feasibility of the multivariate spectroscopic approach as a replacement for expensive and time-consuming conventional chemical methods. Thus, a convenient and feasible method for the quality control of fermentation raw materials for food additives and fine chemicals, especially in CSL, is established.