In this paper, two simple, cost-effective and fast methods for quantification of methotrexate (MTX) in the macromolecular conjugates are presented. The method for analysis of total MTX in preparations was based on absorption spectrophotometry. Validation was performed by measuring absorbance at 372 nm of the sodium bicarbonate solution. Curve describing drug concentration against absorption had a linear character in the range of 1.204-40.13 microM. The reproducibility and precision of method was 0.1558 to 3.086%. The recovery of the method was between 99.56 and 104.7%. The limit of quantitation method was 1.050 microM. The method for free MTX determination was based on size exclusion chromatography and UV-VIS detection at the wavelength of 302 nm. Superdex Peptide column (150 x 4.6 mm) and a mobile phase 0.1 M sodium bicarbonate with a flow rate of 0.4 mL/min was used. In the free drug determination method, the curve had a linear character in the range of 2.006-200.6 microM. The reproducibility and precision of method was 0.3761 to 2.452%. The recovery of the method was between 93.18 and 104.5%. The limit of quantitation method was 0.9203 microM.