Background/Objectives: African Swine Fever (ASF) is one of the most significant infectious diseases affecting both domestic pig and wild boar populations, leading to substantial economic and biosanitary consequences. In Europe, disease management relies on stringent biosecurity measures and surveillance through diagnosis, highlighting the urgent need for an effective and safe vaccine for ASF control. In this context, the VACDIVA project has generated several promising vaccine candidates, including those with the EP153R gene deleted and replaced by the eGFP reporter gene. Methods: In this study, pEP153R and eGFP proteins were produced using recombinant technology and demonstrated their antigenicity and DIVA capability through indirect ELISA. Additionally, a prototype serological DIVA test was designed and developed. The assay is based on the detection of antibodies against both DIVA antigens and the well-established immunogenic p72 protein. Results: This preliminary DIVA diagnostic assay complements vaccine candidates based on a genotype II ASFV strain, featuring the deletion of the EP153R gene and/or the insertion of the eGFP reporter gene, exemplified by the Lv17/WB/Rie1-∆CD vaccine candidate. Conclusions: This approach could potentially improve surveillance during prospective vaccination campaigns.