This method comparison demonstrated equivalent analytical and diagnostic performance of IndiMix JOE with intype IC-RNA as an alternative real-time polymerase chain reaction (RT-PCR) chemistry compared to the National Animal Health Laboratory Network (NAHLN) influenza A virus (IAV) RT-PCR surveillance assay using AgPath-ID One-Step RT-PCR Reagents in avian swabs and tissues and bovine milk. In avian samples, IndiMix JOE with intype IC-RNA using a fast reduced-volume protocol had comparable results to the NAHLN reference RT-PCR with and without intype IC-RNA using standard NAHLN thermocycling conditions. A six-way comparison of RT-PCR chemistries and exogenous internal controls in milk samples illustrated equivalent mean CT values (ANOVA, p = 0.9938). Additional experiments in avian swabs and tissues, bovine milk and semen samples resulted in comparable analytical sensitivity in limits of detection (LOD), linearity (R2 > 0.977), and PCR efficiencies, and no significant differences in mean CT values (ANOVA, p > 0.05). Diagnostic performance showed 100% agreement across field and spiked sample matrices compared to the NAHLN reference method. The study supports the use of IndiMix JOE and intype IC-RNA as an alternative, with practical improvements relevant to surveillance workflows including enhanced testing flexibility, efficiency, outbreak response capacity, and reduced time to results.