A liquid chromatography coupled with ion trap-time of flight mass spectrometry (LC-IT-TOF MS/MS) profiling method was developed for the analysis of the phenolic constituents in Dendrobium. Six different species of Dendrobium were analyzed. Eighteen phenolic constituents in the samples were tentatively identified based on their mass spectra and related literatures. Moscatilin and gigantol were chosen as potential quality control targets of all species of Dendrobium. A high performance liquid chromatography (HPLC) method was developed for quantitative analysis of moscatilin and gigantol in Dendrobium. The extraction condition for moscatilin and gigantol was optimized by the response surface methodology (RSM). The linearity of the method is more than 0.9998 and the precision is within 3% for all the two analytes. The limits of detection (LOD) were 0.18 mg/L and 0.09 mg/L respectively. Recoveries were 97.1% and 101.4% respectively. The study might provide a basis for the quality control of Dendrobium.