A rapid, sensitive method based on neutral desorption (ND) reactive extractive electrospray ionization mass spectrometry (EESI-MS) has been established for the selective quantitative detection of diethylene glycol (DEG) in toothpaste products without any sample pretreatment. The sensitivity and specificity of DEG detection were enhanced by implementing selective ion/molecule reactions in the EESI process, featuring the EESI mass spectra with the characteristic signals of DEG. The method provided a low limit of detection (LOD) (approximately 0.00002%, weight percent of DEG in toothpaste), reasonable recovery (97.6-102.4%), and acceptable relative standard deviations (RSD < 8%, n = 8) for direct measuring of DEG in the spiked toothpaste samples. Trace amounts of DEG in commercial toothpaste products have been quantitatively detected without any sample manipulation. The results demonstrate that nonvolatile compounds such as DEG can be sensitively liberated using the neutral gas beam for quantitative detection from the extremely viscous toothpaste containing solid nanoparticles, showing that ND-EESI-MS is a useful tool for the rapid characterization of highly complex and/or viscous samples at molecular levels.