Monkeypox virus (MPXV) is a zoonotic infection that can be spread human-to-human (Lim et al., 2023). It has two main clades, which can be further divided into subclades: clade one (I), which is divided into clade Ia and Ib, and clade two (II), which is divided into clade IIa and IIb (Government of Canada, 2024). These clades were previously known as the Congo Basin and Central African clade, and the West African clade, based on regions of endemicity (Government of Canada, Nov. 18, 2024, World Health Organization, 2024, Vakaniaki et al., 2024). While few MPXV cases were previously seen outside of sub-Saharan Africa, multi-country outbreaks occurred in 2022 and 2024; largely driven by elimination of smallpox vaccination programs and viral adaptation enabling human-to-human transmission (Kmiec and Kirchhoff, 2022, Government of Canada, 2024). In May 2022, clade IIb MPXV cases were seen in large numbers across multiple non-endemic countries including Canada (Government of Canada, 2024, World Health Organization, 2024)). Furthermore, in September 2022, three MPXV cases in California had a significant deletion in the TNF receptor gene (Lab Alert, 2022). At this time, the MPXV detection assay routinely used by many clinical laboratories including the BC Centre for Disease Control Public Health Laboratory (BCCDC PHL) was a qPCR assay targeting TNF receptor gene (G2RG; OPG002) based on the Li et al. (2010) design and multiplexed with human beta-globin (HBG) as an endogenous control. With new deletions in the TNF receptor, these California cases were undetectable by the Li assay, prompting the need for a second MPXV target. GP113 (OPG128) was chosen as it is in the core conserved region of the genome and aligns with the vaccine target and deletion sequences (Desingu et al., 2024, Ndodo et al., 2023, Wang et al., 2022). In 2024, another multi-country MPXV outbreak occurred, this time driven by an emerging clade Ib strain from the Democratic Republic. Although there hasn’t been an outbreak of this clade in Canada (Government of Canada, 2024), clade Ib has SNPs in the GP113 target, specifically the forward primer, that could impact qPCR detection, and which needed to be addressed using redundant primers. The aim of this work was to validate a triplex G2RG/GP113/HBG qPCR assay that could detect all MPXV clades and provide redundancy against emerging genetic variants.