What's in the picture
In the still, two identical characters stand side by side: green, with round ear-like headphones, wide smiling mouths, and small antennae on their heads. Both are wearing yellow shorts and white baby bibs. Their headgear differs: the one on the left has a red cap, the one on the right a dark red patterned headband.
The main difference is on the bib. The one on the left has two cherries drawn on it, the one on the right a single dark berry. This small detail is the viewer's only clue, so the joke is built on how easy it is to miss. The background of tall green plants emphasizes the cartoonish nature of the scene and recalls the series' forest setting.
Origin
The characters appeared in the animated series Luntik and His Friends, which premiered in 2006. The project's creator is considered to be Konstantin Bronzit, and the plot follows a small creature who came to Earth from the Moon, and his friends. Vupsen and Pupsen serve here as twin antagonists who are constantly up to mischief and getting into trouble.
It was precisely the combination of "two identical, and mischievous" that made them convenient subjects for internet humor. The meme isn't tied to any particular episode: viewers discussed the characters in comments, on forums, and in videos about children's cartoons. The exact date the joke first appeared online is hard to establish, but its peak popularity came in the years after the series' release.
How the joke arose
Almost immediately, viewers noticed that the twins look practically identical and differ only by a tiny detail. This gave rise to the question "How do you tell Vupsen from Pupsen?", which became a recurring subject of jokes. Users began posting stills with captions like "Vupsen or Pupsen?", and in response people would offer answers pointing to the cherries and the berry as clues.
Over time, quizzes in the spirit of "Which Luntik character are you?" got added, where the viewer picks a character for themselves. These quizzes picked up the idea of the lookalike pair and turned it into a format of personal identity. The joke became light and recognizable, understandable even to those who'd only seen fragments of the series.
How it's used and why it stuck
The typical use is a comparison between two very similar people, siblings, twins, or pairs of things that get mixed up with one another. The caption "Vupsen or Pupsen?" transfers easily to any situation where a difference matters but is nearly imperceptible. In this way the meme works as an ironic question about similarity.
It also stuck because the characters evoke nostalgia in those who watched Luntik as kids. Jokes about the negative characters sound good-natured, since no one takes them seriously. The twins' duality, their rivalry, and the tiny visual clue create an ideal setup for a game in which anyone can test their own powers of observation.