A precise and sensitive high performance liquid chromatographic (HPLC) assay method was developed and validated for the quantitation of 2-[4-(2-piperidinoethoxy) phenyl]-3-phenyl-(2H)-1-benzo(b)pyran (compound CDRI-85/287) in rat serum. This method, applicable to 0.5 ml volumes of serum, was validated according to GLP guidelines. It involved double extraction of serum samples with a mixture of hexane and iso-propanol (98:2 v/v) at alkaline pH and the use of UV detection at 332 nm. Linearity, precision and accuracy were acceptable (5-200 ng ml-1. The absolute recovery was more than 75% and the lower limit of quantitation was 5 ng ml-1. Freeze-thaw stability studies up to four cycles showed no apparent differences in the calculated spiked concentrations. However, in-process stability evaluation showed the stability of the processed samples lasted up to 85 h.