Reversible protein phosphorylation represents a key regulatory mechanism that triggers essential cellular signaling events. The large-scale characterization of protein phosphorylation in a cell represents, therefore, the objective of many biological studies that aim at elucidating the complex signaling pathways that are involved in the progression and/or regression of a disease. The recent implementation of novel MS detection strategies has significantly advanced the capabilities for interrogating the complex cellular phosphoproteome. Simultaneously, the current advent of miniaturized technologies has clearly demonstrated the superior performance of microfluidic instrumentation for bioanalytical and biological applications that cope with speed, sensitivity and throughput-related demands. This review aims at providing an update on the latest developments regarding the interfacing of microfluidic devices with MS detection for exploring the challenging area of phosphoproteomics.