Secreted phospholipases A₂ (sPLA₂) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA₂s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA₂s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA₂s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA₂ proteins corresponded to the expression of sPLA₂s at the mRNA level. With a few exceptions, the sPLA₂ proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA₂ is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA₂ in the intestine of GIIA sPLA₂-deficient C57BL/6J mice is not compensated by other sPLA₂s under normal conditions. Outside the gastrointestinal tract, sPLA₂s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA₂ proteins in mouse serum and tissues in various experimental settings.