Early and mature biofilm formation in the extremely halophilic euryarchaeon Halobacterium salinarum strain R1 was characterized by SWATH-LC/MS/MS. Using a simple surfactant-assisted protein solubilization protocol and one-dimensional ultrahigh performance nanoflow chromatography on the front end, 63.2% and 58.6% of the predicted Hbt. salinarum R1 proteome could be detected and quantified, respectively. Analysis of biophysical protein properties, functional analysis and pathway mapping indicate that we achieved a comprehensive characterization of the proteome. 60.8% of quantified proteins (or 34.5% of the predicted proteome) exhibited significant abundance changes between planktonic and biofilm states, demonstrating that haloarchaeal biofilm formation represents a profound “lifestyle change” on the molecular level. Taken together our results and analysis constitute the first comprehensive study to track the molecular changes from planktonic cells to initial and mature archaeal biofilms on the proteome level. Proteins exemplifying different protein expression level profiles were selected, and their corresponding gene transcripts targeted by qRT-PCR to test the feasibility of establishing rapid PCR-based assays for archaeal biofilm formation.