Flos Lonicerae, referred to the flower buds of several medicinal Lonicera species, is a commonly used traditional Chinese herbal medicine. A multi-component-assay quality control method, using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI/TOF MS), has been developed for the simultaneous identification and quantification of 32 bioactive compounds in Flos Lonicerae. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.002–0.089 and 0.006–0.355 μg/ml, respectively. All calibration curves showed good linear regression (r2 ≥ 0.99) within the test ranges. The overall intra- and inter-day precisions of analytes were less than 3.47% for peak area and 0.38% for retention time. The recoveries were from 85.4% to 101.6%. The validated method was applied to assay of 32 compounds in 8 medicinal Lonicera species. Furthermore, six unknown chromatographic peaks were tentatively characterized. It was demonstrated that the HPLC-ESI/TOF MS method was suitable for quality control of Lonicera species, owing to the advantages of accurate mass analysis, resolving power, enhanced selectivity and high sensitivity.