The　human　MUS81-EME1&2　complexes　are　structure-selective　endonucleases　that　play　important　roles　in　DNA　damage　repair.　Here,　we　describe　a　protocol　to　determine　the　endonuclease　activities　of　MUS81-EME1&2　complexes　toward　various　DNA　structures.　We　co-express　MUS81　with　EME1　or　EME2　and　purify　the　complexes　with　high　purity,　and　determine　their　activities　on　the　cleavages　of　3ˊ　flaps,　5ˊ　flaps,　nicked　double-stranded　DNAs,　and　Holliday　junctions.　This　protocol　can　also　be　used　for　the　determination　of　substrate　preferences　of　other　structure-selective　endonucleases.　For　complete　details　on　the　use　and　execution　of　this　protocol,　please　refer　to　Hua　et　al.　(2022).　©　2022　The　Author(s)