Background: Interference refers to learned associations and established behaviors “interfering” with response to new material. It forms a core pillar of executive functions, which are commonly affected in Parkinson’s disease (PD). Cognitive interference test (CIT) forms part of a smartphone application designed for ambulatory assessment in PD. Objective: The aims of this study were to establish that CIT could effectively demonstrate interference and would perform comparably to the Stroop Color-Word Test Victoria version (VST) despite PD-related motor impairment. Methods: Ninety-nine patients with PD were recruited. Initial evaluation included CIT, VST, Montreal cognitive assessment (MOCA), and Movement Disorders Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS-III). A group of patients underwent repeat assessment within 2 weeks. Thirty-four healthy controls were recruited for comparison. Results: Patients’ mean age was 66.2 years, disease duration was 8.7 years, on-state MDS-UPDRS-III was 22, and MOCA total score was 27. CIT effectively generated interference, whereby the total time taken to complete the incongruent task was 20% longer compared to that of the baseline task. CIT key test items demonstrated convergent validity to VST (r = 0.478–0.644, p < 0.0001) and satisfactory repeatability (intraclass correlation coefficient 0.46–0.808, p ≤ 0.0002). Performance on key CIT test parameters deteriorated with increasing age (r = 0.225–0.478, p < 0.01) and MDS-UPDRS-III total score (r = 0.354–0.481, p < 0.0001). When compared to controls and patients with less motor impairment, patients MDS-UPDRS-III > 30 took longer to complete CIT and VST and had lower MOCA-attention sub-score, implying that the degree of motor impairment could not be the sole explanation for reduced CIT performance. Conclusions: We established that despite motor impairment, the novel approach of using smartphone technology to test interference in PD patients is feasible.