Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI‐TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI‐TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.