Because allopregnanolone and derivatives represent biologically active molecules, in this letter, we present the synthesis of a new bioconjugate steroid pyridinium salt derived from allopregnanolone in three steps. The key steps involve the formation of the hydrazone intermediate, followed by condensation with bromoacetyl bromide and subsequent coupling with pyridine to generate the pyridinium bromide salt. The new bioconjugate steroid pyridinium salt, 4, was fully characterized by proton and carbon nuclear magnetic resonance (¹H and ¹³C NMR) spectroscopy, mass spectrometry (MS), and Fourier transform infrared spectroscopy (FTIR). ¹H-NMR analysis revealed the presence of a dynamic rotameric mixture in a 7:3 ratio of Z/E amide conformers, which were identified by a 2D NOESY experiment.