When do I ask for compensation for use of my work?

Agree with above - no discount. They almost certainly won't pass on the savings to their client. But if you feel like you want to honor the spirit of your license, as your work is ultimately being used for a non-profit purpose - charge what you would normally charge for a commercial use of your work, then buy yourself a nice [dinner/bottle of wine/t-shirt] and make a personal donation back to the non-profit with whatever is left over.

re: michaelh's comment - this is a lot more usage than stock images though, they're looking to create an adaptation of OP's comic. I'd charge a fair bit more than $200. You'll have to weigh up the size of the ad agency and their client to balance out exactly where you end up.

If you feel strange about taking the money, want to support the NPO, or perhaps have a significant tax liability, another option would be to make an in-kind gift of the rights for fair market value. They get the work, there is recognition that your work has value, and you get a deduction to help offset your tax liability. Talk to an accountant if this is something you want to pursue, but I will say that we had a photographer at the last place I worked who did this specifically because it helped offset his self-employment tax liability.

What they said. If it helps ease your conscience any in indirectly charging a non-profit for an awareness campaign, remember that if they hadn't found your comic, the ad agency would have commissioned an artist to make something like it -- any money you make from it was budgeted for long before your name entered into the equation.

The less you charge the advertising agency, the more profit they put in their own pockets and any discount you may be considering is very unlikely to translate to savings for the final non-profit customer. Your client is not the non-profit, it's the for-profit advertising agency. Charge a fair price with no discounts.

Speaking as an agency employee, they are okay with paying, and whether they directly pass on expenses or indirectly price services to cover their expenses shouldn't change what you do. $200 is about what some stock images would cost them, if it helps.

Agreed. Is the advertising company getting paid to do this project, or is it donated labor? If they're using your work instead of paying a freelancer or an in-house employee, then it's not non-commercial work. If they intend to use your work and pass the savings on to the client and *you are OK with that,* make sure this is very clear. If they are billing and just using your work in the paid piece, they should be paying you. From one robot hug drawing cartoonist to another, don't let ad people who get paid more than you take advantage of your work. Sometimes it is ok to let people use your stuff for free, but this is a case where it's worth asking who is benefitting. (edit: On re-read, they are getting paid to do the work and I got a bit ahead of myself there!)

People who fund non-profits know that the non-profit will have marketing and design expenses. It's built into the non-profit's budget. Don't be exorbitant, but charge an hourly rate x how long it took to draw the comic. More than a couple hundred dollars and I would raise my eyebrows.

Just to add to the easing of your guilt, I worked for a non-profit and we worked with creative agencies, paid for work, did advertising, etc. Yeah it was nice if something was donated, and sometimes we got discounts or two-fers, but we had a budget.