Many factors have been found to have important effect on the retention of solutes in high performance liquid chromatography (HPLC). (These factors include stationary phase type and size of particles, mobile phase composition, flow rate, column dimensions, etc. The first factor is considered the most effective in the separation of sample component in HPLC. This has been the reason for synthesis of wide. variaties of stationary phases for the separation of different kinds : of samples. This also explain the availability of so many HPLC columns suitable for any application In this work, new mixed stationary phase was exploited for the separation of some amino acids such as alanine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, phenylalanine, and tryptophan. Mixed octadecylsilane(ODS) and cyanopropyl(CN) stationary phases have been investigated at different proprtions from 0-100% weight ratio, in order to find out the best weight ratio to separate these amino acids effectively and in a short analysis time . This study was based on LAUB and PURNEEL equation :- tR mix = tR ODS X WODS + tR CN X WcN In which; tR mix , tR ODS ,and tR CN are the retention times in mixed, ODS, and CN column, respectively. WODS and WCN are the weight fraction of ODS and CN stationary phases, respectively. We have found that the best ratio was 40% ODS and 60% CN that gives the most efficient separation, using 0.01M KH2PO4 adjusted to pH=5 as mobile phase and detection at 220 nm. The new mixed stationary phase has been packed into a stainless steel column. This has been clone using down-flow packing technique. The chromatographic performance of the new packed column was characterized. The plate number, height equivalent to theoretical plates, capacity factors, and selectivity factors were me —../e^ by chromatographic analysis of amino acid standards. The average va'.ue of plate number N, and plate heights H, were 14535 and 0.00:54 cm. respectively, while the capacity factors K', and separation factors a, were ranged from 1.30-15.98 and 1.12-2.91, respectively. Calibration curves for amino acids analyzed with this new column were linear from their detection limits to at least 50 ppm with-a correlation coefficient ranged from 0.9990-0.9997. The detection limits were ranged from 0.05-0.10 ppm at a signal to noise ratio of three or more.