The innate immune system is the body’s first line of defense against infection. A critical component of the innate immune response—conserved from plants to humans— is the sensing of pathogenic or danger signals by sensors called inflammasomes. In mammals, inflammasomes once activated form large signaling complexes that alert other immune cells to mount a larger response against infection or pathology. In particular, inflammasomes recruit and activate the cysteine protease CASP1 to cleave and mature cytokines IL-1 and IL-18, and GSDMD which forms membrane pores causing an inflammatory form of cell death known as pyroptosis. Understanding the role and regulation of inflammasomes in pyroptosis will not only contribute to our fundamental understanding of the immune system, but could also lead to the development of new therapeutics for infection, autoimmunity, and cancer. Here I present three stories that provide insight into the workings of the NLRP1 (nucleotide binding, leucine rich repeat, pyrin domain containing 1) inflammasome in rodents and its homolog CARD8 (caspase activation and recruitment domain containing 8) in humans, as well as the role of proteases in the activation and regulation of these related sensors. In the first study, we reveal the mechanism underlying rodent NLRP1B activation by anthrax lethal factor (LF) protease using a genome-wide CRISPR screen and cellular biochemical analysis. In particular, we describe how LF protease, known to cleave NLRP1B, creates a neo-N-terminus which is detected by N-end rule machinery to degrade the N-terminus. The N-end rule protein degradation pathway recognizes unstable new N-termini generated in cells, followed by polyubiquitination and eventual proteasomal degradation. Since NLRP1B has a unique domain that can autoproteolyze and form two cleaved fragments that stay non-covalently bound to each other, the proteasomal degradation machinery targets the N-terminal fragment, freeing the C-terminal fragment to recruit CASP1 and cause pyroptosis. This work provides a unifying model of NLRP1 activation. The second study investigates the role of cytosolic peptidases DPP8 and DPP9 as novel checkpoints of mouse NLRP1B activation. We show that inhibition of DPP8/9 in macrophages activates the NLRP1B inflammasome, but without direct cleavage of the protein. This work also introduces a small molecule inhibitor of DPP8/9, Val-boroPro (VbP), as a new tool to activate the NLRP1B and related inflammasomes. In the third study, we aim to differentially activate the related inflammasome, CARD8. We find that inhibition of the enzyme prolidase (PEPD) sensitizes immune cells to pyroptosis by CARD8 activation. Altogether, this thesis elucidates the molecular mechanisms of NLRP1 and CARD8 activation and establishes new tools to activate these inflammasomes. More broadly, this work provides a framework for identifying the pathogenic and danger signals that are sensed by inflammasomes, and paves the way for inflammasome-based therapeutics for the treatment of infection, autoimmunity and cancer.