The peculiar isomer-selective reduction of (α)-hydroxyiminophosphonates (oxime isomers) into (α)-hydroxyaminophosphonate (hydroxylamine) derivatives is presented. A library of 16 (α)-hydroxyiminophosphonates is prepared and studied via a unique multifaceted approach involving the interplay of NMR, XRD, MS, IM-MS and computational chemistry techniques. The combination of NMR, XRD and HPLC enables the seamless separation, identification and quantification of the oxime isomers (E/Z). Tandem MS (MS/MS) enables the determination of the fragmentation patterns for both isomers. Collision energy breakdown curves highlight the order of apparition of the fragments as well as their related energy of fragmentation, demonstrating that the strength of the C–P bond in the Z isomers is much weaker than in the E isomers. Computational chemistry demonstrates that favorable protonation site is isomer-dependent with the phosphonate moiety being the favorable protonation site for the E isomers, while protonation occurs preferentially on the amino moiety for Z isomers regardless of the phosphite source. The combination of these various methods led an unprecedented level of characterization of oxime isomers, providing a better uderstanding of the isomer-dependent behavior of (α)-hydroxyiminophosphonates.