MALDI-TOF MS has recently been proposed as an accurate tool for arthropod identification, including ticks. In this study, we evaluate and confirm the ability of MALDI-TOF MS, to identify different tick species collected in Cameroon, considering other lines of evidence (morphology and molecular). A total of 1483 adult ticks were collected from cattle in five distinct sites in the Western Highland of Cameroon. Because of engorged status and/or absence of some morphological criteria, some Ixodes spp. and Rhipicephalus spp. were identified to the genus level only. Among those, 944 ticks (543 males and 401 females) were selected for the current work. They were classified into 5 genera and 11 species: Rhipicephalus (Boophilus) microplus (31.7%), Rhipicephalus lunulatus (26%), Amblyomma variegatum (23%), Rhipicephalus sanguineus s.l. (4.8%), of Haemaphysalis leachi group (4.6%), Hyalomma truncatum (2.6%), Hyalomma rufipes (1.7%), Rhipicephalus muhsamae (1.1%), Rhipicephalus (Boophilus) annulatus (0.6%), Rhipicephalus (Boophilus) decoloratus (0.3%), Ixodes rasus (0.1%), Ixodes spp. (0.2%) and Rhipicephalus spp. (3.3%). Tick legs were subjected to MALDI-TOF MS analyzes, and the spectra of 929 (98.4%) specimens were of good quality. Analysis of these spectra provided intra-species reproducibility and interspecies specificity of MS profiles obtained from the different species. Our in-house MALDI-TOF MS arthropod database was upgraded with spectra from 44 specimens of 10 different tick species. Blind testing of good quality spectra revealed that 99% agreed with the morphological identification. Of these, 96.9% had log score values (LSVs) between 1.73 and 2.57. MALDI-TOF MS also allowed to correct the morphological misidentification of 7 ticks, and to identify 32 engorged ticks that were not morphologically identifiable to the species level. This study supports MALDI-TOF MS as a reliable tool for tick identification and provides new data on tick species identification in Cameroon.