The use of high performance thin-layer chromatography (HPTLC) in combination with high resolution time of flight mass spectrometry (MS) for the detection, identification and imaging (HPTLC/MSI) of ecdysteroids (insect moulting hormones) present in a number of plant extracts obtained from members of the Silene family is demonstrated. DESI is shown to be a convenient method for the recovery of these polar polyhydroxylated steroids from the silica gel of the HPTLC plate for subsequent MS detection and imaging. The incorporation of an ion mobility separation (IMS) to the system to give HPTLC/IMS/ MS provided additional drift time data which enabled more confident identification. Using HPTLC/DESI/IMS/MS, a range of ecdysteroids were detected and characterized in extracts of S. otitis, S nutans, S. maritime, S. viridiflora and S. fimbriata.