The herbicide is the part of the pesticide, these herbicide are normally used to control the weeds in the cultivation period. The quality of the herbicide has to be effective and the degradation should be fast. After the usage, the soil fertility has to be maintained without any environmental chain changes. There are many herbicides are available to control weeds or unwanted plants in the cultivation processes. Many of the herbicides are used to control the grass or very small plants, which has a very week in the structure. The Picloram herbicide used to control especially the plant which has strong stem and branches. The usage is unique and this herbicide has to be used carefully to maintain the required cultivated plants during the plantation or cultivation period. There are few herbicides are available to control wood plants. The specialty of the picloram herbicide will not have any action on the grass. Fluroxypyr is another molecule being used as a herbicide in the cultivation industry. The chemical nature of this molecule is similar to that of the picloram molecule. The Fluroxypyr is consisting of the pyridine molecule basically. Fluroxypyr has fluorine, chlorine, amine and an ether arrangement in its structure. This Fluroxypyr is used to control woody plant which has broadleaf. This molecule also has the electronegative structural annulments in it has more penetration power through roots of the plants. Mostly this Fluroxypyr is being used to control weeds in the place where the cultivations are normally carried out. These Picloram + Fluroxypyr molecules were separated through a mobile phase consisting of the mixture of acetonitrile and water (fortified with 0.5% of formic acid) in the ratio of 40:60 v/v. The separation was achieved through the Qualisil BDS C8 (250 x 4, 5μ) column with the flow rate as 1.5 ml/min with the detection at 254 nm. These method parameters were loaded in the Shimadzu HPLC (model: LC-2030). The LC solution Shimadzu software was used for all the calculations in this analytical method validation analysis. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the identification and quantifications of these molecules interims of validation parameters viz., separation, system suitability, System Precision and linearity in a simple HPLC analysis.