I'd punctuate it as:

"It's been, what, twenty-five years since I've seen you?".

I don't think the "what" is, in itself, a question here. As dicitonary.reference.com says, it's an interjection:

interjection

 

23. (used in exclamatory expressions, often followed by a question):

 

What, no salt?

Also the "twenty five years" isn't a question on it's own either. What we have is a statement posed as question: "It's been twenty-five years since I've seen you?" with the interjection "what" inserted in the middle.

To mark the pauses in speech either side of what, I'd use commas.

I'd punctuate it as:

"It's been, what, twenty-five years since I've seen you?".

I don't think the "what" is, in itself, a question here. As dicitonary.reference.com says, it's an interjection:

interjection

23. (used in exclamatory expressions, often followed by a question):

What, no salt?

Also the "twenty five years" isn't a question on it's own either. What we have is a statement posed as question: "It's been twenty-five years since I've seen you?" with the interjection "what" inserted in the middle.

To mark the pauses in speech either side of what, I'd use commas.