Hard ticks (Acari: Ixodidae) have been historically identified by morphological methods which require highly specialized expertise and more recently by DNA-based molecular assays that involve high costs. Although both approaches provide complementary data for tick identification, each method has limitations which restrict their use on large-scale settings such as regional or national tick surveillance programs. To overcome those obstacles, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced as a cost-efficient method for the identification of various organisms, as it balances performance, speed, and high data output. Here we describe the use of this technology to validate the distinction of two closely related Dermacentor tick species based on the development of the first nationwide MALDI-TOF MS reference database described to date. The dataset obtained from this protein-based approach confirms that tick specimens collected from United States regions west of the Rocky Mountains and identified previously as Dermacentor variabilis are the recently described species, Dermacentor similis. Therefore, we propose that this integrative taxonomic tool can facilitate vector and vector-borne pathogen surveillance programs in the United States and elsewhere.