The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1β) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.