Monkeypox virus (MPXV) has caused two Public Health Emergencies of International Concern since 2022, with co-circulation of distinct clades (Ia, Ib, IIa, IIb) that differ in virulence and transmission patterns. Rapid discrimination of these clades is critical for outbreak control and clinical management. Here, we developed and validated a two-tube multiplex real-time quantitative PCR (qPCR) assay targeting the H3L gene, designed to first distinguish clade I from clade II, followed by subtyping into clades Ia, Ib, IIa, and IIb in separate reactions. The assay demonstrated high sensitivity, with detection limits of 100 copies/reaction for clade Ia, 10 copies/reaction for clade Ib, 10 copies/reaction for clade IIa, and 1 copy/reaction for clade IIb using standard plasmids. Excellent specificity was confirmed by the absence of cross-reactivity with nucleic acids from other human pathogens, including vaccinia virus Tiantan strain (VTT), human adenovirus (HAdV), getah virus (GETV), chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), dengue virus (DENV), influenza A virus (IAV), influenza B virus (IBV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The assay exhibited high repeatability, with intra-assay coefficients of variation (CVs) ranging from 0.09 to 1.88% and inter-assay CVs from 0.23 to 2.68%. Clinical evaluation using 48 skin samples from infected crab-eating monkeys or SCID mice showed 100% concordance with singleplex qPCR for clade IIb detection. For clades Ia, Ib, and IIa, clinical validation was not feasible due to sample unavailability, and their performance was analytically confirmed using recombinant plasmids. Importantly, the clade assignment of the 48 animal specimens was not confirmed by sequencing, which represents a limitation of the current study. Furthermore, these specimens were derived from animal models rather than human patients; thus, the results represent preclinical validation, and formal clinical validation in human specimens from endemic regions is warranted. This two-tube multiplex qPCR assay provides a rapid, sensitive, and reliable tool for the analytical discrimination of all four major MPXV clades, with preliminary clinical validation for clade IIb. Its ability to distinguish emerging clades makes it a valuable asset for clinical diagnosis, epidemiological surveillance, and future outbreak preparedness, although further clinical validation in endemic regions is required.