The updated version of EU GMP Annex 1 mentions the possibility of introducing alternative methods for microbiological contamination control, which can provide results as real-time methods directly during the measurement. This is intended to detect possible contamination in critical process environments more quickly and thus reduce the risk to product quality. Microbiological real-time methods in air are based on the excitation of certain cell-based substances using laser radiation and the subsequent measurement of the induced fluorescence. In addition to conventional cleanroom qualification, the method is also used in pharmaceutical Grade A filling in controlled isolator environments. When implementing the measurement method, a number of important points must be taken into account due to the measurement conditions within the critical area.