A shielded-hydrophobic-phase (SHP) HPLC method for the determination of residual methotrexate in recombinant protein biopharmaceuticals was validated. The method requires no removal of protein or other prior sample c lean-up and detects quantities of methotrexate as low as 2.5 ng in the presence of up to 25 mg/ml of protein. Methotrexate was fully resolved from a recombinant IgG1 monoclonal antibody and associated matrix components. Accuracy was demonstrated by measuring s piked recoveries at the limit of quantitation (found 90-120% recovery with R.S.D.s =<10%). Other validation parameters studied included range, precision, ruggedness, robustness and stability of s piked samples.