This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / A Fistful of Dynamite
If you were in Juan's place when he rode that carriage in the beginning of the film, you'd be pretty hurt by the other people talking about you like you weren't there, like you weren't human.
The discovery that Juan's entire band of outlaws, including all of his sons, have been brutally massacred. And of course John's death.
The execution scene. John's moment of horror, too, once he finds that Dr. Villega is a traitor.
Juan begins the story only interested in riches and getting that one big score. Later in the story after the death of his family, he finds himself with a bag of valuables worth a small fortune. They may as well be made from tin for all Juan cares. John's silent acknowledgement of the situation is equally heartbreaking.
The looks shared between Juan and John while hiding out in the cargo carriage of a train. John catches Juan wiping away his tears, and tries to look away because he knows that it'll make Juan ashamed of himself and just feel worse.
John's last flashback of Ireland, slowly fading away as he's dying.