Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high‐resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision‐induced dissociation (CID), higher‐energy collisional dissociation (HCD), and pulsed Q collision‐induced dissociation (PQD) MS² fragmentation, with MS² CID followed by MS³ and MS⁴ fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.