Nucleoid-associated proteins (NAPs) are key factors in maintaining chromosomal DNA integrity, gene regulation, DNA replication, and DNA repair. A recently identified mycobacterial NAP, NapMin (NapM) from Mycobacterium smegmatis, was a subject of the study. NapM modulates the expression of about one-third of M. smegmatis genes. Among these genes, those involved in cell envelope biosynthesis and cell division comprise a particular group specific to NapM. A bottom-up proteomics approach was utilized to define NapM interactors on protein level. For this purpose, a NapM pull-down was first performed from M. smegmatis cell lysate on antiFlagTag resin. Proteins associated with the resin were then identified via mass spectrometry. A WT cell lysate sample was parallelly processed in the exact same manner as background control.