New methods for studying the complexity of multiple PTMs in functional proteomics are required to understand cell signaling processes. In this study, a multiplexing 2DE-based approach is introduced for parallel analysis of the redox-, phospho-, and total-proteome. This triplexing approach uses spectrally distinct fluorophores, is not matrix-specific and requires relatively low sample amounts with applicability to any cell/tissue type. This methodology was applied for the study of Target of Rapamycin (TOR) regulating pathways in Chlamydomonas reinhardtii. With emerging research demonstrating a complex yet unclear relationship between TOR kinase, autophagy, and lipid metabolism, rapamycin treatment was used to induce TOR inhibition in C. reinhardtii and redox-, phospho- and total proteome changes were assessed using the triplexing approach. We identified a total of 68 spot abundance changes in response to TOR inhibition which provide a basis for understanding this highly conserved, master regulator in algae.