For each campaign you can enable "Unsubscription Management".
This will add an "unsubscribe" link to each email you send. If a recipient clicks on that link they will be added to your unsubscription list and shown the unsubscription page (which you can customize in your account settings). Once an email is on your unsubscription list any future attempt to send to that email address will be prevented and your campaign dashboard will show "Previously unsubscribed". The email address of a unsubscriber will not be checked when you upload a source file, only once the campaign is being sent.
You can manually add or remove email addresses from your unsubscription list.