Curcumin and its derivatives generally display favorable cytotoxic activities against a number of cancer cell types. We focus our rational antineoplastic drug design program on curcumin analogues containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore. Favorable outcomes from pharmacological screens of this series demanded further pharmacokinetic evaluations to determine their suitability as effective compounds in vivo. To allow such evaluations and to provide a general, sensitive, rapid and simple method for the analysis of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold, we developed an HPLC method with ultraviolet detection for their detection in various biological matrices of a relevant preclinical species, i.e. the rat. Our HPLC method is specific for the analysis of many members in this series in rat blood, plasma, serum and hepatic microsomes following liquid-liquid extraction with TBME (1:30, v/v). The assay procedure involves chromatographic separation on a Zorbax-Eclipse C-18 column under isocratic conditions with the mobile phase consisting of acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in different ratios depending upon the compound. The method was validated for NC 2083 in rat serum and rat liver microsomes, a potential lead compound, to demonstrate its applicability. The standard curve was linear (r 2 ≥ 0.997) from 50 to 5000 ng/mL. Intra-and interday precision and accuracy of the method were within USFDA specified limits. The stability of NC 2083 was established in an auto-injector, on bench-top, during freeze-thaw cycles and longterm stability at −80 °C for 40 days. The method is suitable for a number of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold with divergent log P values with only minor adjustments in the buffer to acetonitrile ratio of the mobile phase.