Nowadays biodiesel (fatty acid methyl ester, FAME) has been becoming an important issue as a desired alternative of energy products because of non-toxic, biodegradable properties, and lower exhaust emissions. During esterification of fatty acids or transesterification of oils and fats with short chain alcohols by the alkali-catalyzed methanolysis, FAME and unrefined glycerol are generated. Quantification of glycerol as a by-product is important because of a determinant of biodiesel quality. However, the glycerol analysis by gas chromatography (GC) method has laborious works with sample preparation, long time and cost of sample analysis. Thus, there is a need to analyze glycerol more simply. Herein we demonstrate that the colorimetric assay for glycerol analysis conducted by UV-vis spectrophotometer at the wavelength 617 nm whose peak is maximum intensity of malachite green, resulting in the red-shift occurred proportionally as a function of glycerol amount. Thus, it is considered the solvent media for malachite green fading for biodiesel production: (1) water, (2) MeOH, and (3) EtOH. The resulting findings show that the peak intensity at 617 nm in glycerol-malachite green mixture had a relationship between glycerol concentration and degree of peak shift as increase in pure glycerol concentration approximately at pH 7.0. However, when it was measured the unrefined glycerol concentration by diluting and adjusting with water to buffer (pH 7.0), it was not observed the absorption peak at 617 nm because of impurities and OH ions. In case of glycerol from biodiesel production factories, glycerol concentration could be successfully measured.