Sirtuins are a class of enzymes with important functions in regulation aging, metabolism, and genome stability. They were originally known as nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases. However, recently it has been discovered that certain sirtuins with weak deacetylase activity also hydrolyze novel acyl lysine modifications. These findings indicate that other sirtuins with weak deacetylase activity may also possess novel activities on unknown protein posttranslational modifications. Analytical methods that can help to identify new activity of sirtuins and new acyl lysine modifications are thus needed. Here we describe a sensitive method that uses (32)P-labeled NAD and thin-layer chromatography to detect sirtuin-catalyzed deacylation reactions. This method can help to discover new acyl lysine modifications that can be removed by novel sirtuin activities.