Healthcare providers may soon have a precise tool to determine whether someone's mpox antibodies came from natural infection or vaccination, addressing a critical gap in monitoring both individual immunity and population-level outbreak responses. The ability to distinguish these antibody sources has been hampered by the genetic similarity between monkeypox and vaccinia viruses used in vaccines. A newly validated blood test analyzes antibody responses to five specific monkeypox virus proteins alongside their vaccinia virus counterparts, creating distinct antibody fingerprints for each type of exposure. Testing across 257 individuals—including 26 with confirmed mpox infections, 52 JYNNEOS vaccine recipients, and 179 unexposed controls—the assay achieved over 96% accuracy in detecting exposure to either virus. Individual protein markers like VACV D8L and MPXV B6R proved excellent for confirming any orthopoxvirus exposure but couldn't differentiate the source. The breakthrough came from comparing antibody ratios between corresponding monkeypox and vaccinia proteins, which successfully separated natural infection from vaccination with high precision. This diagnostic advance arrives as public health agencies grapple with tracking immunity levels during ongoing mpox outbreaks while expanding vaccination programs. The test's ability to correlate with neutralizing antibody levels—particularly for the B6R/B5R and E8L/D8L protein pairs—suggests it could also help assess protective immunity duration. However, the study's relatively small infection cohort and focus on recent exposures means longer-term antibody patterns remain unclear. For epidemiologists and clinicians, this represents a significant step toward evidence-based immunity assessment rather than relying solely on exposure history, which patients may not accurately recall or report.