The spinal subarachnoid space (SSAS) is vital for neural performance. Although models of spinal diseases and trauma are used frequently, no methods exist to obtain high-resolution myelograms in rodents. Thereby, our aim was to explore the feasibility of obtaining high-resolution micro-CT myelograms of rats by contrast-enhanced dual-energy (DE) and single-energy (SE) digital subtraction. Micro-CT contrast-enhanced DE and SE imaging protocols were implemented with live adult rats (total of 18 animals). For each protocol, contrast agents based on iodine (Iomeron® 400 and Fenestra® VC) and gold nanoparticles (AuroVist™ 15 nm) were tested. For DE, images at low- and high-energy settings were acquired after contrast injection; for SE, one image was acquired before and the other after contrast injection. Post-processing consisted of region of interest selection, image registration, weighted subtraction, and longitudinal alignment. High-resolution myelograms were obtained with contrast-enhanced digital subtraction protocols. After qualitative and quantitative (contrast-to-noise ratio) analyses, we found that the SE acquisition protocol with Iomeron® 400 provides the best images. 3D contour renderings allowed visualization of SSAS and identification of some anatomical structures within it. This in vivo study shows the potential of SE contrast-enhanced myelography for imaging SSAS in rat. This approach yields high-resolution 3D images without interference from adjacent anatomical structures, providing an innovative tool for further assessment of studies involving rat SSAS.