Kdo2-lipid A and Kdo2-monophosphoryl lipid A (Kdo2-MPLA) connect Gram-negative outer-membrane biochemistry with the analytical design of lipopolysaccharide-derived vaccine-adjuvant candidates. Their characterization is demanding because acyl-chain number, acyl-chain position, phosphate state, Kdo substitution, and ion adduction jointly shape reversed-phase retention and tandem mass-spectrometric fragmentation. This study establishes a retention–fragmentation biosynthetic fingerprint for eight structurally assigned Kdo2-lipid A derivatives from engineered Escherichia coli analyzed by triethylamine-assisted reversed-phase LC–MS/MS. Each species was encoded by acylation class, phosphate number, total acyl carbon count, secondary-acyl-chain identity, primary-chain length, precursor-adduct state, and diagnostic MS/MS evidence. A constrained two-parameter retention rule based on total acyl carbon number and phosphate count explained the observed retention ladder with high internal consistency (R2 = 0.992; root-mean-square error = 0.158 min; leave-one-out mean absolute error = 0.19 min). Matched structural contrasts showed that diphosphorylation advanced elution by 0.15–0.16 min within tetra- and hexa-acylated scaffolds, whereas replacement of C13:0(3-OH) by C14:0(3-OH) delayed elution by 0.24 min in the penta-L series. Diagnostic MS/MS evidence from Kdo/Kdo2 ions, 0, 4A2 cross-ring fragments, phosphate loss, acyl-chain loss, and adduct-dependent suppression of glycosidic cleavage separated MPLA-like, diphosphoryl lipid A (DPLA)-like, and adduct-dominated spectra. The fingerprint distinguishes target MPLA products, residual diphosphorylated species, and primary-chain microheterogeneity, giving a chemically constrained quality-control readout for Kdo2-MPLA engineering.