Sirtuins are class III HDAC proteins capable of hydrolyzing acyl modifications on lysine residues, which has an important function in cellular metabolism and transcriptional control. Sirtuins play a role in both healthy and tumorigenic cells, and thus the development of sirtuin modulators has been of interest in the last decade. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. SIRT2 was initially thought to be a deacetylase, but it has been demonstrated that SIRT2 is also an efficient long chain deacylase. With our recently developed SIRT2 long chain deacylase assay, we have shown that two known inhibitors, suramin and SirReal2, have different inhibitory profiles against substrates containing ε-N-acyllysine modifications of varying length. We have developed a scalable and purification-free synthesis of Ac-ETDK(myristoyl)-AMC, the fluorogenic substrate used in our SIRT2 deacylase assay, facilitating therefore its application in other groups. Furthermore, the strategy presented is suitable for the synthesis of libraries of fluorogenic compounds and is potentially applicable to other peptide sequences.