Inhibitors of the sphingosine-1-phosphate (SIP) degrading enzyme SIP lyase (SPL) may be useful in the therapy of inflammatory diseases by preventing lymphocyte recruitment to diseased tissues. Here we describe a cellular assay for such inhibitors, which takes advantage of the observation that a fraction of the intracellular SIP accumulated in the presence of SPL inhibitors is secreted into the medium of cultured cells. The secreted SIP is then quantified using an SIP-sensitive reporter cell line. In the routine assay protocol, human HEK293T cells are treated with SPL inhibitors in the presence of phosphatase inhibitors and sphingosine; while the phosphatase inhibitors are included to prevent the degradation of SIP secreted from the cells, sphingosine is added as source for intracellular SW that is prone to SPL degradation. The secreted SIP in the supernatant of the cell cultures is then quantified by measuring calcium flux induced in CHO-K1 cells expressing the human S1P(3) receptor. Using this method SPL inhibitors were shown to induce a concentration-dependent increase of extracellular SIP under the conditions used; thus, the assay allows for the ranking of SPL inhibitors according to their potency on living cells. (C) 2013 Elsevier Inc. All rights reserved.