The reference methods for Nocardia identification are based on gene sequencing. These methods are time-consuming and not accessible for all laboratories. Conversely, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is easy to use and widely available in clinical laboratories, but for Nocardia identification, the VITEK®-MS manufacturer recommends a tedious step of colony preparation that is difficult to integrate into a laboratory workflow. This study aimed to evaluate Nocardia identification by MALDI-TOF VITEK®-MS using direct deposit with the VITEK®-PICKMETM pen and a formic acid-based protein extraction directly onto the bacterial smear on a 134 isolates collection; this identification was compared to the results from molecular reference methods. For 81.3% of the isolates, VITEK®-MS delivered an interpretable result. The overall agreement with the reference method was 78.4%. Taking only the species included in the VITEK®-MS in vitro diagnostic V3.2 database into account, the overall agreement was significantly higher, 93.7%. VITEK®-MS rarely misidentified isolates (4/134, 3%). Among the 25 isolates that produced no result with the VITEK®-MS, 18 were expected, as Nocardia species were not included in the VITEK®-MS V3.2 database. A rapid and reliable Nocardia identification using direct deposit by VITEK®-MS is possible by combining the use of the VITEK®-PICKMETM pen and a formic acid-based protein extractiondirectly onto the bacterial smear.