Objective To identify the new designer drugs which are totally unknown and not in the routine testing list by the technologies such as high-resolution mass spectrometry in drug facilitated sexual assault, in order to solve the problem in actual cases. Methods The milky fluid from an actual case was extracted and analyzed using LC-QE, 1H-NMR and GC-MS, respectively. The accurate masses and cluster ions isotope patterns of unknown compound were obtained by LC-QE. The molecular formula was confirmed as C16H12Cl2N2O based on the protons number of 1H-NMR. The isomers diclazepam and 4-chlorodiazepam were separated and detected with GC-MS. Results The new designer benzodiazepine as diclazepam in the milky fluid was identified. The results provided direct evidence for the investigation and qualitative analysis of such cases. Conclusion The combined application of various methods, including LC-QE, 1H-NMR and GC-MS, can be used to detect unknown new psychoactive substances.