This study quantifies the behaviour of uranium isotope ratios (²³⁵U/²³⁸U and ²³⁴U/²³⁸U) with different aqueous sample preparation and separation techniques using multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) measurements and optimization of the MC-ICP-MS measurement protocol to obtain high precision and accuracy for uranium isotope ratios. The obtained results were compared with reference values, and they show that different sample preparation and separation methods influence the uranium isotopic composition and consequently the final results of unknown samples. The analytical procedure with the highest achievable precision and accuracy for uranium isotopes was the combination of coprecipitation with Ca₃(PO₄)₂ and extraction chromatography with precleaned UTEVA resins. The combination of coprecipitation with Fe(OH)₃ and ion exchange chromatography (DOWEX resins) provided the lowest achievable accuracy for uranium isotopes. The results reveal the extent of uranium biases during different physicochemical processing of samples in the range of −254 to 2.60‰ for ²³⁵U/²³⁸U and −299 to 8.90‰ for ²³⁴U/²³⁸U. A combination of sample preparation and measurement protocol optimization is of importance for various applications requiring extensive sample pre-treatment procedures where the ²³³U/²³⁶U double spike method for correcting for mass bias during sample preparation and separation steps is not feasible and to establish a methodology which enables the accurate determination of uranium isotope ratios in low-level concentration samples.