Why did I click that? I knew what would happen because started happening before I clicked it. Oh well, my system needed flushing. 
The death of Mary Gold in the Gumps caused quite a reaction from what I’ve read (I wasn’t around then).
I’ve always teared up at My Dog the Paradox
I don’t have a specific example, but Cyanide & Happiness has some doozies. There’s this series, but they might have worse ones.
I don’t know why exactly they would make it so the robot is somehow homesick despite the fact if we’re putting emotion and logic into it it should realize there’s no realistic way for it to get back home and should instead focus on the job at hand. The fact its mission exceeding 20 times it’s planned should be the thing it’s proud of, not the fact it thinks it did a bad job hence why it’s not coming home.
Because that wouldn’t be sad.
That was actually only half of the story. In the following strips, Calvin & Hobbes go back to where they found the raccoon and see it’s next to a construction site. The raccoon’s burrow (and likely the rest of his family) had been dug up and plowed under.
That’s another one that does me in. Just thinking about it and I’m
I would submit Garfield Minus Garfield for honorable mention.
At first the premise is amusing, but once you’ve gotten past the novelty and shuffled through a few dozen strips, you come to realize that Jon is a deeply troubled person who is clearly suffering from chronic depression and start to feel for him.
Maybe not saddest ever but the xkcd Tatoo gets me misty
One of my favorite animated tales from C & H: Waiting for the Bus.
Not necessarily a tear-jerker, but one that has stayed with me all these years is Non Squiters’ “The Old Man’s Tattoo”. https://mymorningmeditations.files.wordpress.com/2012/04/nonsequitur-holocaust.jpg
What made it even sadder: most people who read it first saw it in the same paper that reported that Schulz had died the previous day.
For that matter, pretty much any comic strip published on May 27, 2000 could qualify as “sad”; quite a few of them had references to Peanuts or Schulz in them, as Schulz was going to be honored at that year’s National Cartoonists Society banquet that night, and, without him finding out, his wife had planned for as many strips as possible to mention him as a surprise (because of the amount of “lead time” needed, the strips were drawn and submitted before he died).
He had several related to his fiance/wife’s cancer diagnosis and treatment that were tear-jerkers. Spirit (from the OP) is the saddest in a jokey way, but these are actually sad.
For saddest animated cartoon, the episode of Futurama with Fry’s dog.
I dunno; “Tattoo” doesn’t get me misty-eyed. It gets me going “Hell yeah, kick that tumor’s… um… tendrils!”.
Lisa Moore’s death in Funky Winkerbean.
A different strip from The Oatmeal: It’s going to be okay.
I never knew that about Gene Roddenberry.