The aim of our research was to perform a comprehensive study of ecdysterone (ECD)-containing dietary supplements (DSs). Two analytical methods were optimised according to the expected concentration of the target compounds: quantitation of ECD by liquid chromatography (LC) coupled to diode array detector (DAD), and limit test for 47 World Anti-Doping Agency prohibited by LC coupled with tandem mass spectrometer (MS/MS). For quantitation of ECD, the method was fully validated with outstanding performance characteristics (LOD: 35 µg·g−1, LOQ: 115 µg·g−1, CV% < 5%), resulting in significantly lower LOD and shorter runtime than published previously. For limit tests, a chromatographic method was developed to obtain excellent separation, while MS/MS parameters were optimised to allow the lowest possible reporting limit (RL: 0.6–10 ng·g−1 or mL−1). Twenty-one ecdysterone-containing DSs from ten brands were analysed. In all cases, the measured ECD content was much lower than labelled, and 20% of the samples contained a prohibited substance. The concentration of ecdysterone and contaminations varied randomly from batch to batch. The developed methods help to prevent the use of contaminated or useless DSs.