In comparison to single-molecule localization microscopy (SMLM) of proteins located on the cell surface, SMLM of proteins within the nucleus requires fluorescent tags that are generally brighter, longer-lasting and (in the case of fluorescent ligands) membrane-permeable. Although fluorescent ligands typically have superior optical qualities compared to those of fluorescent protein (FP) tags, making them ideal candidates for nuclear SMLM, unlike FPs, they are unable to be expressed in cells as a fusion protein with the protein of interest. Self-labelling tags, on the other hand (e.g., HaloTag, SNAP-tag, and CLIP-tag), combine the ability to express a protein tag (e.g., HaloTag protein) as a fusion protein, with the enhanced optical qualities of externally added fluorescent dyes (e.g., dye-conjugated HaloTag ligand which covalently binds to the HaloTag protein). Excitation of only a subset of fluorescently labelled molecules is essential for achieving single-molecule resolution, and therefore SMLM of nuclear proteins requires a microscope setup capable of penetrating the nucleus with a thin beam that is powerful enough to produce an appropriate signal-to-noise ratio. Highly Inclined and Laminated Optical sheet (HILO) microscopy uses an oblique angle (just below that of Total Internal Reflection Fluorescence (TIRF)) to excite fluorescently labelled molecules within a thin, highly inclined plane, the signal of which can be amplified using a high-power TIRF filter. This chapter describes the following protocols: (i) seeding of cells into glass-bottom imaging dishes, (ii) transfection of cells with a HaloTag-tagged nuclear protein of interest, (iii) incubation of transfected cells with a fluorescent nuclear marker and a fluorescent HaloTag ligand, and (iv) single-molecule imaging of the fluorescently labelled HaloTag-tagged protein of interest in the nucleus of live cells using HILO microscopy with a high-powered TIRF filter.