A few studies have investigated the analysis of neonicotinoid insecticides in honey bee royal jelly. An analytical method based on salting-out liquid-liquid extraction assisted with ultrasound followed by liquid chromatography-tandem mass spectrometry was developed for the determination of thiamethoxam and clothianidin in royal jelly. A cleaning step based on freezing before injection to the UHPLC-MS/MS (MRM mode) allowed maximizing the signal-to-noise ratio. The finalized method was validated in line with European legislation. The limits of quantification (LOQ), reached from a test sample of 100 mg royal jelly only, was 0.25 ng/g for both neonicotinoids. The recoveries were close to 100% with relative standard deviation below 13.7%, measured at three concentration levels. The validated method was finally applied to samples of royal jelly commercially available, and showed the presence of thiamethoxam at levels between the limit of determination (LOD = 0.15 ng/g) and LOQ in a few samples.