Holy Water in the Face
The Winchester men have a long history with holy water. They have done their fair share of flinging it at demons. In many cases, it is one Winchester wielding holy water against another Winchester as a test. Here is a look at all those times:
After Dean finds Sam holding Jo at knife point, Dean splashes Sam with holy water to confirm that he is possessed by a demon. Meg, in Sam's meatsuit, escapes and heads to Bobby's house, where Bobby discovers that Sam is possessed when he adds holy water to Sam's beer. Later, after Bobby secures the possessed Sam, Dean splashes Sam again to try to get the demon to talk.
On Gordon Walker's orders, Kubrick and Creedy capture Sam. They splash Sam with water that may or may not be holy water. However, they believe that Sam was helping the demons who opened the Devil's Gate, and they believe that there is something supernatural about Sam, so it may have been holy water.
Dean tests Adam for any demon-ness by swapping out tap water for holy water in the restaurant where they meet.
When Dean discovers that Sam is back from Hell, Sam wants to prove he isn't a monster and since Dean didn't throw any holy water in Sam's face, Sam drinks some holy water laced with salt to prove that he is human.
After Samuel Colt kills some demons, he is suspicious when Sam arrives, and so he splashes Sam with holy water.
Dean returns from Purgatory and when he first meets Sam, Dean tests him with holy water, borax, and silver. Then, when Sam refuses to test Dean, Dean proves it is him by testing himself the same way. Later, Kevin squirts them with borax or holy water, it is not specified, however, Dean states that they are not Leviathans.
When Henry first arrives in 2013, Sam and Dean do not know who he is, so they splash Henry with holy water to make sure that he isn't a demon.
Kevin uses a squirt gun to spray Sam and Dean with holy water. Apparently the holy water doesn't work because the two guys, who appear to be Sam and Dean, are really demons.
In order to get information about the First Blade, Dean and Crowley seek out a hunter named Tara, who had worked with John at one time. Tara decides that Dean must be possessed if he was working with the King of Hell, so she tests him by splashing him with holy water.
So he can get close enough to inject him with purified blood, Sam splashes Dean with holy water. Later, Sam splashes Dean again to check if he is cured.
In order to get the upper hand, Cole splashes Dean because he believes that Dean is a demon.