If you have changed the default workspace file (default.rui for Rhino 5 or later, default.tb for Rhino 4) in any significant way, save it under a new name.
When Rhino 4 is uninstalled, the default workspace file is deleted that wipes out all your customization in an instant. Updating Rhino 4 will not overwrite the default workspace file. In Rhino 5 or later, you will not lose the default workspace file when Rhino is uninstalled or updated, but workspace files may get corrupted on rare occasions. Using the command ToolbarReset will also replace the existing default file, and any customization made to it will be lost as well.
Go to Tools > Toolbar Layout (or Options > Toolbars), highlight the default file in the upper right window, then click File > Save As from the menu and give your changed workspace a new, recognizable name. Now continue to use this file for your work.
Always do all your modifications on your custom workspace. We recommend that you save this file somewhere else as well as a backup (preferably on another computer, like all important documents). Think of how much time it will take you to recreate your file if you lose it.
Installing Rhino updates or ToolbarReset will not affect your personalized workspace file.
Make sure only one instance of Rhino is open when making toolbar/workspace modifications! Once done with your toolbar installations, make sure you use File>Save from the toolbar layout dialog to save your changes! Or, exit Rhino and reopen. Rhino saves toolbar changes automatically on exit. If you have multiple instances of Rhino open, the last one to close will overwrite the previous one, so you might lose your changes.
The workspace file only contains information related to toolbars, not to other Rhino options. For a complete overview of where all the Rhino options are stored and how to back them up or transfer them, see this page.
The above info applies to Rhino 6 and earlier. Some of it may change for future versions.