I've searched for a bit, but not found an answer. What is the best/easiest way to determine if a 24 bit audio file is truly 24 bits vs a 16 bit file that has been padded to 24 bits?

Adobe Audition in Amplitude Statistics shows "actual bit depth". As far as I remember, when I did a 24-bit recording with a USB Audigy, it showed up as having actual bit depth of 16-bit. Audigy (and Extigy) were marketed as 24-bit cards, and they had 24-bit converters, but there was an obligatory 16-bit processor in the signal path and the lowest 8 bites were truncated.

I do use Audition, and actually saw that but didn't know if it would recognize padded 16 bit files or not. I will create a test file and check that out.

WaveLab's bit meter can indicate similarly when configured to the "Intuitive mode". With a 16-bit file padded to 24-bit, the bit indicators will only light up to 16, indicating meaningless data after the 16th bit.

@pawelq I just tested this in Audition and it does correctly report a 16 bit file that has been padded to 24 bit as 16 bit. Thanks for pointing this out to me.