ABSTRACTInfectious disease diagnostics often depend on costly serological testing with poor sensitivity, low specificity, and long turnaround time. Here, we demonstrate proof of the principle for simultaneous detection of two tick-borne pathogens from a single test sample using barcoded magnetic bead technology on the BioCode 2500 system. Specific primer sets complementary to the conserved genes ofAnaplasma phagocytophilumandBorrelia burgdorferiwere used in PCR amplification of the target, followed by the hybridization of the resulting biotinylated PCR products with specific probes tethered to the barcoded magnetic beads for simultaneous detection, using a fluorophore with high quantum yield. The assay has an extremely high signal to background ratio, with a limit of detection (LOD) of 2.81 50% tissue culture infection dose (TCID50)/mL and 1 CFU/mL forA. phagocytophilumandB. burgdorferi, respectively. The observed LOD for gene blocks was 1.8 copies/reaction for both the pathogens. The assay demonstrated 100% positive and negative agreement on performance evaluation using patient specimens and blood samples spiked with 1 × LOD of pathogen stock. No cross-reactivity was observed with other related tick-borne pathogens and genomic DNA of human, cattle, and canine origin. The assay can be upgraded to a sensitive and cost-effective multiplex diagnostic approach that can simultaneously detect multiple clinically important tick-borne pathogens in a single sample with a short turnaround time.IMPORTANCEThe low pathogen load in the tick-borne disease test samples and the lack of highly sensitive multiplex diagnostic approaches have impacted diagnosis during clinical testing and limited surveillance studies to gauge prior insight about the prevalence of tick-borne infections in a geographical area. This article demonstrates proof of the principle for simultaneous detection of two important tick-borne pathogens from a single test sample using digital barcoded magnetic bead technology. Using a fluorophore of high quantum yield, the diagnostic approach showed high sensitivity and specificity. The LOD was 1.8 genome copies per reaction for bothA. phagocytophilumandB. burgdorferi. The assay can be upgraded for the detection of all clinically important tick-borne pathogens from a single patient sample with high sensitivity and specificity. The assay can provide a diagnostic answer to the clinician in a short turnaround time to facilitate speedy therapeutic intervention to infected patients and implement public health measures to prevent community spread.