Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus ( Boophilus ) microplus , two Amblyomma varanensis , seven Dermacentor auratus , one Dermacentor compactus and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys , Anaplasma phagocytophilum , Anaplasma marginale , Ehrlichia rustica , Babesia vogeli , Theileria sinensis , and Theileria orientalis were detected in 25 ticks. Co-infection by A . phagocytophilum and T . sinensis was found in one Rh . ( B ) microplus .