Stone fruit production is immensely threatened by viral diseases worldwide. In recent years, the spread of stone fruit viruses like apple chlorotic leaf spot virus (ACLSV), prunus necrotic ringspot virus (PNRSV), plum pox virus (PPV), prune dwarf virus (PDV), and cherry virus A (CVA) has been reported to cause devastating losses worldwide. In this study, three viruses that infect stone fruits, i.e., apple chlorotic leaf spot virus (ACLSV), cherry virus A (CVA), and prunus necrotic ringspot virus (PNRSV), were simultaneously detected and differentiated using a multiplex reverse transcription polymerase chain reaction assay. The optimized parameters were the annealing temperature, extension duration, and primer concentration. The multiplex RT-PCR assay detected ACLSV, CVA, and PNRSV, yielding specific amplicons of 677 bp, 1089 bp, and 346 bp, respectively. The assay demonstrated a detection limit of 10 pg of total RNA for ACLSV, 1 pg for PNRSV, and 100 pg for CVA. The devised technique detected all three viruses in single or multiple infections of the stone fruits (apricot, cherry, peach, plum, and nectarine) after the multiplex RT-PCR assay was validated using samples collected in the field. This study developed a multiplex RT-PCR assay that can be further utilized for indexing and diagnosing stone fruit viruses in field-grown populations.