To evaluate the validity of the newly developed Oral Hygiene Ability Instrument (OHAI), created to assess the cause of any inability of older adults to perform oral hygiene, and to revise the instrument based on the results. Good oral hygiene is among the most important prophylactic measures for oral health. This applies especially to older adults, among whom risk factors and physical and cognitive barriers are more common and can hamper oral hygiene. The OHAI contains (I) an interview, (II) clinical examination, (III) observation of oral hygiene activities and a summarising part. In the study, 149 older adults in three groups participated: stroke, cognitive disorder and general dental patients. Inclusion criteria were to be ≥65 years old, have at least one tooth and to manage oral hygiene without assistance. For criterion validity, sensitivity and specificity were calculated using eight reference instruments. To determine construct validity, we used known group validity, factor analysis and Rasch analysis. The criterion and construct validity of the OHAI were found to be acceptable to good. In the stroke group, balance and fine motor skills were assessed to affect oral hygiene most; in the cognitive disorder group, it was balance, coordination, spatial ability and cognitive functions. Analyses revealed that one item had no added value and that some response options were not optimal. The OHAI proved to be valid for the group it is intended for, with only minor revisions needed, resulting in a 32-item instrument. The OHAI could be a valuable person-centred tool in prophylactic work with older adults with failing oral hygiene.