Background: Fast and reliable determination of antibiotic resistance of bacteria causing septicemia is crucial for adapting an antiinfective therapy. A new method for fast detection of β-lactam resistances in bacteria has been developed based on co-incubation of ß-lactamase producing bacteria with a ß-lactam antibiotic directly after isolation from positive blood cultures and subsequent detection hydrolysis of the ß-lactam ring by MALDI-TOF. Methods: We applied this MALDI-TOF MS based method on a subset of 40 positive blood cultures from hospitalized patients suffering from E. coli sepsis. Specimen were collected directly from positive blood culture bottles, enriched by a short sample preparation and incubated in an ampicillin solution for 90 minutes. Supernatants were directly spotted onto a MALDI target and spectra were acquired on a bench top mass spectrometer “microflex LT/SH”. Spectra were analyzed using a novel software algorithm which calculated the ratio of he hydrolyzed and non-hydrolyzed form of the antibiotic (log RQ). Results: By MALDI-TOF MS the samples could be assigned to two groups. One group showing a mean log RQ above 0.5 and the other group showing a mean log RQ below 0. Isolates with a mean log RQ below 0 were defined to be susceptible to ampicillin, all isolates above 0.5 as resistant. The application of these simple criteria led to 15 ampicillin sensitive and 25 ampicillin resistant isolates. For validation E-tests for ampicillin were performed in each case and interpreted according to CLSI rules. Comparison between the E-tests as the gold standard and the new MALDI-TOF MS method gave a 100% accordance. Conclusion: The MADDI-TOF MS based detection of β -lactam resistance in E. coli directly from positive blood cultures is feasible. This enzymatic activity test directly from positive blood cultures may be extended to the detection of other resistance determinants such as ESBL and carbapenemases.