Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification.