This study was undertaken to evaluate the utility of matrix-assisted laser desorption ionization–time of flight mass spectrometry with the Vitek MS Plus system for identifying
Mycobacterium abscessus
subspecies in order to facilitate more rapid and appropriate therapy. A total of 175 clinical
M. abscessus
strains were identified by whole-genome sequencing analysis: 139
Mycobacterium abscessus
subsp.
abscessus
and 36
Mycobacterium abscessus
subsp.
massiliense
. The research-use-only (RUO) Saramis Knowledge Base database v.4.12 was modified accordingly by adding 40
M. abscessus
subsp.
abscessus
and 19
M. abscessus
subsp.
massiliense
reference spectra to construct subspecies SuperSpectra. A blind test, used to validate the remaining 116 isolates, yielded 99.1% (
n
= 115) reliability and only 0.9% (
n
= 1) error for subspecies identification. Among the two subspecies SuperSpectra, two specific peaks were found for
M. abscessus
subsp.
abscessus
and four specific peaks were found for
M. abscessus
subsp.
massiliense
. Our study is the first to report differential peaks 3,354.4
m
/
z
and 6,711.1
m
/
z
, which were specific for
M. abscessus
subsp.
massiliense
. Our research demonstrates the capacity of the Vitek MS RUO Saramis Knowledge Base database to identify
M. abscessus
at the subspecies level. Moreover, it validates the potential ease and accuracy with which it can be incorporated into the IVD system for the identification of
M. abscessus
subspecies.