The Mediterranean citrus industry is threatened by the devastating citrus tristeza disease caused by the Citrus tristeza virus (CTV). The presence of severe strains and efficient insect vectors further increases the risk. In this scenario, the early detection and characterization of severe CTV strains is deemed crucial, requiring effective RNA extraction and analysis methods. In this regard, this study undertook a comparative analysis of RNA yield and purity, sensitivity, processing time, ease-of-use, and cost of three extraction methods: semi-automated magnetic bead (MB), silica capture (SC) and membrane printing (MP). The last method was also subjected to optimization in terms of printed membrane size, incubation conditions, explant type, and potential synergy with magnetic beads. The MB method demonstrated exceptional efficiency with high RNA yield and purity, fast processing, and user-friendliness, albeit at a relatively higher cost. Relating to MP optimization, increasing printed membrane size showed potential improvements, but integration with MB method reduced its efficiency. The use of MB and enhanced membrane printing methods holds great promise for advancing CTV monitoring and potentially benefiting the Mediterranean citrus industry.