Iron and Mn deficiency are the most common micronutrient deficiencies in soybean [Glycine max (L.) Merr.] in the United States, although deficiencies in Zn and Cu have also been observed in the field. This study was conducted to assess the utility of leaf fluorescence and reflectance as a rapid means of detecting Mn, Zn, Fe, and Cu deficiency, and to determine whether spectral methods could be used to derive critical concentration levels for Mn, Fe, Zn, and Cu. ‘Bragg’ soybean plants were raised as seedlings in aerated, chelator-buffered nutrient solutions in a growth chamber. Manganese, Zn, Cu, and Fe were supplied over a wide range of concentrations to induce a range of deficiency symptoms. Chlorophyll concentrations and reflectance and fluorescence characteristics of the youngest fully mature leaf were measured periodically. Reflectance characteristics responded to decreases in leaf total chlorophyll concentration, while fluorescence characteristics responded to changes in electron transport associated with Mn, Zn, Cu, or Fe deficiency. Critical concentration ranges estimated from spectral measures were 9 to 10 mg kg−1 for Mn, 12 to 18 mg kg−1 for Zn, 45 to 50 mg kg−1 for Fe, and 1.0 to 1.5 mg kg−1 for Cu. The spectral measures used in this study were useful in detecting Mn, Zn, Fe, and Cu deficiencies and may provide a rapid, nondestructive method for the detection of these deficiencies in the growth chamber, greenhouse, or field. The spectral measures were also useful for establishing critical levels for Mn, Zn, Fe, and Cu under controlled conditions.