Antimicrobial resistance is a global threat to human health and welfare, food safety, and environmental health. The rapid detection and quantification of antimicrobial resistance are important for both infectious disease control and public health threat assessment. Technologies such as flow cytometry can provide clinicians with the early information, they need for appropriate antibiotic treatment. At the same time, cytometry platforms facilitate the measurement of antibiotic-resistant bacteria in environments impacted by human activities, enabling assessment of their impact on watersheds and soils. This review focuses on the latest applications of flow cytometry for the detection of pathogens and antibiotic-resistant bacteria in both clinical and environmental samples. Novel antimicrobial susceptibility testing frameworks embedding flow cytometry assays can contribute to the implementation of global antimicrobial resistance surveillance systems that are needed for science-based decisions and actions.