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<i>Xylella fastidiosa</i> (<i>Xf</i>) is a Gram-negative plant bacterium that causes severe diseases affecting several economically important crops in many countries. To achieve early detection of the pathogen, a droplet digital PCR (ddPCR)-based approach was used to detect the bacterium at low concentrations in different plant species and insect vectors. In this study, we implemented the reaction conditions of a previously developed ddPCR assay, and we validated its use to detect <i>Xf</i> in insect vectors as well as in a broader list of host species. More specifically, the sensitivity and accuracy of the protocol were assessed by testing five plant matrices (<i>Olea europaea</i>, <i>Nerium oleander</i>, <i>Vitis vinifera</i>, <i>Citrus sinensis</i>, and <i>Prunus dulcis</i>), and for the first time, the insect vector (<i>Philaenus spumarius</i>), was either naturally infected or artificially spiked with bacterial suspension at known concentrations. The lowest concentrations detected by ddPCR were 5 ag/µL of bacterial DNA and 1.00 × 10² CFU/mL of bacterial cells. Both techniques showed a high degree of linearity, with R² values ranging from 0.9905 to 0.9995 and from 0.9726 to 0.9977, respectively, for qPCR and ddPCR. Under our conditions, ddPCR showed greater analytical sensitivity than qPCR for <i>O. europea</i>, <i>C. sinensis</i>, and <i>N. oleander</i>. Overall, the results demonstrated that the validated ddPCR assay enables the absolute quantification of <i>Xf</i> target sequences with high accuracy compared with the qPCR assay, and can support experimental research programs and the official controls, particularly when doubtful or inconclusive results are recorded by qPCR.