I've never actually written assembly professionally, but the understanding of what's going on at the CPU level has been invaluable. You could probably run FreeDOS(http://www.freedos.org/) in a VM, looks like it comes with a couple different assemblers.
Hideously outdated, and has practically no real world application any more, but is conversely an incredibly lucid, explanatory, and beatifully written as a guide to 16-bit Assembly language on the IBM (and compatible) PC - Guides you through first examinations of registers and memory through to creating a low-level hard disk editor.
Pop open DosBox, download MASM, and see what you can break!
Currently going for $3 on Amazon - worth a pop for that price if only for a cursory look!