Two accurate analytical methods were developed for the resolution and analysis of a binary mixture of acemetacin (ACE) and chlorzoxazone (CLZ). The first method depends on chromatographic separation of ACE and CLZ using a Shimadzu VP-ODS column. Mobile phase containing 0.075 M sodium dodecyl sulphate, 0.3% triethylamine, 10% n-propanol in 0.02 M orthophosphoric acid of pH 5.5 was pumped at a flow rate of 1 mL/min. with UV detection at 210 nm. The method was further extended to the determination of acemetacin (ACE) and chlorzoxazone (CLZ) in spiked human plasma without prior extraction. For the second method, it depends on measuring the amplitudes of the first derivative spectra at 281.75 nm for ACE and 242.53 nm for CLZ.