The rapid determination of steroidal hormones and their contents in royal jelly by molecular imprinting and high performance liquid chromatography-mass spectrometry (HPLC-MS) was reviewed. The research progress of liquid-liquid extraction, solid phase extraction, QuECHERS and other pretreatment methods, as well as HPLC-MS, gas chromatography-mass spectrometry (GC-MS), radioimmunoassay and enzyme-linked immunosorbent assay were introduced. The advantages and disadvantages of various pretreatment methods and analytical methods were analyzed and compared. Finally, the feasibility of using molecular imprinting technology and HPLC-MS to detect steroid hormones in royal jelly was prospected.