High-affinity aptamers for important signal transduction proteins, i.e. Cdc42-GTP, p21-activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) alpha were successfully selected in the low micro- to nanomolar range using non-systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naive DNA library. In the non-SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non-equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non-SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (K-d) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non-SELEX for the selection of numerous unique sequences with high selectivity.