Aim: Current analytical tools lack the required capacity to reduce the complexity of the plasma proteome and identify low-level proteins of clinical interest. Hence, the need to develop a fractionation approach to provide adequate throughput for a clinical study and minimize the loss and improve the detection of low abundance proteins. Materials & methods: We present the development of an analytical platform that combines the depletion of 12 high abundance proteins and multi-lectin affinity chromatography (12P-M-LAC) fractionation. Results & conclusion: We validated the highly specific, stable and robust 12P-M-LAC platform using human plasma. An improved enrichment of low abundance proteins and glycoproteins with minimum sample loss was achieved demonstrating the suitability of this platform in future biomarker discovery studies.