Solution 1:

There are two places for LaunchAgents to be called from:

  • /Library/LaunchAgents - this launches a process at login for every user
  • ~/Library/LaunchAgents - this launches a process at login for a specific user.

The Spotify startup plist is found in the latter; the com.spotify.webhelper.plist allows you to open Spotify via the web.

Issue the command launchctl list | grep -i spotify and you should find (up to) two plists that are loaded:

$ launchctl list | grep spotify
-       0   com.spotify.client.startuphelper
9087    0   com.spotify.webhelper

To unload them, you can uncheck the option in Advanced Settings (Command,)

enter image description here

Or...

You can issue the command:

$ launchctl unload com.spotify.client.startuphelper.plist

Solution 2:

There are several ways that programs can automatically start in macOS:

First check "Login Items" which are found in System Preferences after clicking the "Users & Groups" icons. I understand that you have already checked here and did not find Spotify.

Another possibility is that Spotify have been set to open at login from the dock. You can right-click the Spotify icon in the dock, and then in the Options menu make sure that "Open at login" is not enabled.

Similarly if you have left Spotify running when you power down the Mac then by default the app and open windows will be opened again after rebooting. You can avoid this by ensuring that you close Spotify before logging out or rebooting, or change the setting in general by clicking the "General" icon System Preferences and making sure there's a checkmark in "Close windows when quitting an app".

A third possibility is that it is possible to create configuration files for launchd that will automatically start programs at boot or login. You'll find these configurations files in:

~/Library/LaunchAgents/
/Library/LaunchAgents/
/Library/LaunchDaemons/

Note that the ~ characters means your user's home folder.

A final possiblity is that applications in macOS can come with a "shared helper application". Essentially this application would provide some background service to one or more actual applications from that developer. Some use that helper application to later launch their actual application.

The helper application is located within the application's bundle in the Contents/Library/LoginItems folder. For Spotify that is usually in the following location:

/Applications/Spotify.app/Contents/Library/LoginItems/StartUpHelper.app

You can prevent that from autostarting Spotify by closing the helper app, or making sure that it cannot start (for example by removing the executable bit on the actual executable: /Applications/Spotify.app/Contents/Library/LoginItems/StartUpHelper.app/Contents/MacOS/StartUpHelper)