Spectrophotometric, coloriaetric and differential pulse polarographic “DPP” techniques were adopted for microdetermination of methotrexate in pure form and in pharmaceutical preparations. The spectrophotometric method was based on measuring the light absorbance at 303 nm in 0.1 N NaCH. The colorimetric method was based on coupling the diazotised methotrexate with 8-hydroxy quinoline in alkaline medium and measuring the colour developed at 430 nm. The three proposed techniques were compared with the E.P. 1980. method. The DPP method was further used for methotrexste determination in presence of some biological fluids.