An in vitro method for detecting an enzyme of a microorganism which is resistant to 3 rd generation cephalosporins, from a biological sample, said enzyme to be detected being chosen from the group formed by extended spectrum β-lactamases, cephalosporinases and carbapenemases, said method comprising the steps consisting of: a1) concentrating the microorganisms present in the biological sample, optionally after a a0) culture step of the microorganisms; b1) suspending the microorganisms concentrated at step a1) in a solution comprising at least one chromogenic substrate capable of releasing a chromophore after hydrolysis by the enzyme to be detected, said chromogenic substrate being the compound HMRZ-86 ( -7-[2-(aminothiazol-4-yl)-(z)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid thrifluoroacetate, E-isomer) or a salt thereof; c1) detecting potential release of the chromophore obtained at step b1); the release of the chromophore detected at step c1) indicating the presence of the enzyme to be detected and said process further comprising, when the biological sample is a sample containing blood or red blood cells: - a step of lysing red blood cells present in the biological sample before the suspending step b1) or before the concentration step a1), and/or - a step a') of preparing the biological sample before the microorganisms concentration step a1), the step a') comprising:: (i) agglutination of the red blood cells, and (ii) separation of the agglutinated red blood cells from the microorganisms present in the sample. a1) concentrating the microorganisms present in the biological sample, optionally after a a0) culture step of the microorganisms; b1) suspending the microorganisms concentrated at step a1) in a solution comprising at least one chromogenic substrate capable of releasing a chromophore after hydrolysis by the enzyme to be detected, said chromogenic substrate being the compound HMRZ-86 ( -7-[2-(aminothiazol-4-yl)-(z)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid thrifluoroacetate, E-isomer) or a salt thereof; c1) detecting potential release of the chromophore obtained at step b1); the release of the chromophore detected at step c1) indicating the presence of the enzyme to be detected and said process further comprising, when the biological sample is a sample containing blood or red blood cells: - a step of lysing red blood cells present in the biological sample before the suspending step b1) or before the concentration step a1), and/or - a step a') of preparing the biological sample before the microorganisms concentration step a1), the step a') comprising:: (i) agglutination of the red blood cells, and (ii) separation of the agglutinated red blood cells from the microorganisms present in the sample. - a step of lysing red blood cells present in the biological sample before the suspending step b1) or before the concentration step a1), and/or - a step a') of preparing the biological sample before the microorganisms concentration step a1), the step a') comprising:: (i) agglutination of the red blood cells, and (ii) separation of the agglutinated red blood cells from the microorganisms present in the sample. (i) agglutination of the red blood cells, and (ii) separation of the agglutinated red blood cells from the microorganisms present in the sample.