As part of the N-glycan processing pathway, Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan₅GlcNAc₂ . The product of this hydrolysis (GlcNAcMan₃GlcNAc₂) is the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII reduce the production of complex N-glycans and are therefore potential cancer therapeutics. Despite many studies targeting the inhibition of GMII, potent and selective inhibitors GMII over other alpha-mannosidases remain elusive, and α-mannosidosis-like symptoms due to inhibition of lysosomal alpha-mannosidase are therefore a major concern. It is in this context that we sought to design a fluorescence polarization screen for new GMII inhibitors. We synthesized both manno-epi-cyclophellitol epoxide and aziridine and demonstrated covalent modification and time-dependent inhibition of Drosophila melanogaster GMII (dGMII) with these inhibitors. The manno-epi-cyclophellitol aziridine was then used as a scaffold to design a fluorescent α-mannosidase activity based probe, which was implemented in a fluorescence polarization based screen for dGMII inhibitors. We identified 7 previously unknown inhibitors of dGMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. This revealed previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective inhibitors.