New stationary phase for high performance liquid chromatography has been prepared by the reaction of Neutral red solution with Weakly acidic cation exchanger resin . The stability of the new attached functional group on the resin was studied against different solvents such as benzene, ethanol, acetonitrile, acetone, methanol, chloroform, and petroleum ether as well as NaOH and HCl solutions. The new prepared resin was found stable and no depletion of the Neutral red moiety from the resin was noticed with the above materials The FTIR analysis has configured the attachment of Neutral red on the resin as well as the physical appearance of the new resins. The new stationary phase has been packed into stainless steel column. The pKa value for new resins has been calculated. The pKa for Weakly acidic – Neutral red resins were 7.15. The chromatographic performance of the packed column was characterized. The number of plate numbers N, height equivalent of a theoretical plates H, capacity factors Ḱ, selectivity factors α, and peaks asymmetry, and Resolution RRsR were measured by analyzing different analytes on the new columns using different mobile phase compositions and flow rates. These parameters were measured from analysis of some amino acids such as Histidine, Phenylalanine, Tryptophan, and Tyrosine.The analysis of amino acids using Weakly acidic – Neutral red column has performed using %10 acetonitrile in buffer phosphate as mobile phase. The average value of plate numbers N was (316.59) . The plate heights H was averaged (0.085) cm , capacity factors Ḱ were ranged from (1.8 – 2.86). Separation factors α were ranged from (0.21-1.17), and peak asymmetry value were range from (0.99-1.09). Resolution RRsR were ranged from (0.93 -3.77). The analysis of amino acids using a new stationary phase has performed using buffer phosphate as mobile phase . The average value of plate numbers N was (234.79) , plate heights H was (0.107)cm , capacity factors Ḱ were ranged from (1.13-4.73) , separation factors α were ranged from (0.23-1.22) , peak asymmetry value were range from (1.10-1.18) and resolution were (0.93). R.S.D were ranged from(0.04 -1)% , recovery were ranged from (93.8-97.12)% , The relative errors were ranged from (-2.88 - -6.2) % Calibration curves for all analyzed compound were linear from their detection limits to at least 50 ppm with correlation coefficient ranged from(0.9991- 0.9996).The detection limits were ranged from(0.05-0.25) ppm at signal to noise ratio of three or more.