AbstractTandem mass spectrometric methods for the identification of 19‐nortestosterone (estr‐4‐en‐17β‐3‐one) are described and evaluated. The fragmentation reactions of the heptafluorobutyryl (HFB) and pentafluorobenzyloxime heptafluorobutyryl (PFBOHFB) ester derivatives of 19‐nortestosterone in particular were studied for the purpose to select characteristic ions. The HFB ester was analyzed by collisionly activated dissociation (CAD) following electron impact in order to fragment the steroid nucleus. Cleavage of the A‐ring, i.e. the ring containing the keto function, was prominent. The formation of A‐, A/B‐ and D‐ring fragments was also typical for this type of derivative. The PFBOHFB ester was formed to create a derivative, which could capture electrons and be analyzed in the electron‐capture negative chemical ionization (ECNCI) mode. Besides fragmentations originating in the groups coupled to the steroid by derivatization, no prominent steroid nucleus fragmentations were observed by CAD following ECNCI. Accordingly, of both methods only CAD following EI of the HFB derivative of 19‐nortestosterone provided a characteristic MS/MS procedure for the identification of 19‐nortestosterone.