Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.