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Aflatoxin B₁ (AFB₁) is one of the most toxic mycotoxins. One of the producers of AFB₁ is <i>Aspergillus flavus</i>. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of <i>A. flavus</i> based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB₁ identification by scraping from TLC plates, <i>A. flavus</i> mycelium, nutrient media around <i>A. flavus</i> growth, and finally direct AFB₁ identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB₁ in the samples of aflatoxin-producing strains of <i>A. flavus</i> and vice versa; AFB₁ was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing <i>A. flavus</i> strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB₁-producing and non-producing strains of <i>A. flavus</i>. Statistical and cluster analysis divided AFB₁-producing and non-producing strains of <i>A. flavus</i> into two monasteries. The results indicate that it is possible to distinguish between AFB₁ producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB₁-producing and non-producing strains of <i>A. flavus,</i> the entire mass spectrum database identification of aflatoxigenic <i>A. flavus</i> strains can be even faster and cheaper, without the need to identify the toxin itself.