Ligand-targeted drugs (LTDs) such as antibody-drug conjugates (ADCs) are currently attracting great attention as an alternative class of therapeutics to conventional chemotherapy for the clinical treatment of cancer. The linker is one of important factors determining the efficacy and toxicity of LTDs. The linker for LTDs should have enough stability during blood circulation, effectively release the payload, and leave no polar moieties in the released payload. However, the drug release activity and plasma stability of cleavable linkers are generally evaluated by complex and sophisticated in vivo techniques containing LC-MS, and the designing of new clinically applicable linkers remains a challenge. In this work, leucine aminopeptidase (LAP)-responsive fluorescent probes were designed as a simple preliminary model to verify whether a peptidase-responsive fluorescent probe can be used as a facile tool for the development of cleavable linkers although LAP is an exopeptidase and can't be a real target for cleavable linkers. LAP-responsive fluorescent probes were prepared by conjugation of a leucine to several xanthene fluorophores through a few linkages with a p-aminobenzyl spacer. The stability tests, kinetic study and live cell imaging of LAP-responsive activatable fluorescent probes demonstrated that the chemical stability and intrinsic activity of the linker for the release of drug can be easily evaluated by a fluorogenic assay. The ex vivo plasma stability test using mice suggested that an enzyme-responsive activatable fluorescent probe can be used as a feasible platform to evaluate the plasma stability of cleavable linkers during blood circulation.