Abstract: Royal jelly is a popular nutritional supplement, its quality directly affects consumer’s health. In this work, solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for determination of amitraz, N-2,4-dimethyl phenyl-N-methylformamidine (DMPF), N-(2,4-dimethylphenyl) formamide (DMF) and 2,4-dimethylaniline (DMA) in royal jelly. The protein of royal jelly was precipitated, and the extracts were dissolved and distilled with ammonia solution and ammonia of acetonitrile solution. The supernatant solution was cleaned up with solid phase extraction column of N-Al₂O₃ cartridges and eluted with ammonia of acetonitrile solution into a glass culture tube, the eluate evaporated to dryness and reconstituted in acetonitrile solution, and then filtered through nylon membrane into a glass LC vial for LC-MS/MS analysis. The solution was separated by Agilent Eclipse XDB-C18 column (150 mm×4.6 mm×5 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring (MRM) mode under positive electrospray ionization (ESI⁺). Isotopes dilution internal standard method or external standard method were used to determine the residue contents in sample. The limits of quantification (LOQs, S/N=10) of amitraz, DMPF, DMF and DMA are 0.05, 0.5, 5.0 and 0.5 μg/kg, respectively. Single-laboratory method validation data were determined, the calibration standard curves concentration are 0, 5.0, 10, 100, 200, 300 μg/kg and correlation coefficients are more than 0.997. Appropriate amounts of the standard target compounds were spiked into the “blank” royal jelly and fixed at four final concentration levels of 5.0, 10, 100 and 200 μg/kg, the recoveries and relative standard deviations (RSDs) using spiked matrix calibration standard curves are 50.5%-110% and 0.8%15.0%, respectively. This method is convenient, rapid and effective, it is suitable for determination and confirmation of amitraz and its metabolites in royal jelly sample, and in line with the regulations of European Union (EU).