The routine analysis of phosphoproteomics needs effective and specific enrichment methods. The strong cation exchange chromatography (SCX) separation conditions, including the buffer solution and the separation gradient, were studied to enrich the phosphopeptides in a short time. Firstly, the tryptic peptides of standard protein mixtures (bovine alpha-casein and beta-casein, chicken egg ovalbumin) were used to optimize the SCX strategy. Then, this method was applied to the complicated samples, the tryptic peptides from yeast. The experimental results showed that the phosphopeptides were eluted before 30 min in the optimized SCX systems with less interference from nonphosphopeptides. Phosphopeptides were successfully separated in a short time, and the ion signals corresponding to phosphopeptides were accordingly increased during mass spectrometric analysis. It was indicated that the optimized SCX separation system could be used as a simple enrichment method for the separation of phosphopeptides, especially for phosphopeptides in the complex real samples, in a short time, and to increase the relative abundance of phosphopeptides in the detection fraction. This study provides the pragmatic technique for phosphoproteomics analysis on a large-scale.