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Pineapple is an economically important tropical fruit crop, but the lack of adequate planting material limits its productivity. A range of micropropagation protocols has been developed over the years to address this shortfall. Still, the final stage of micropropagation, i.e. acclimatisation, remains a challenge as pineapple plantlets grow very slowly. Several studies have been conducted focusing on this phase and attempting to improve plantlet growth and establishment, which requires tools for the non-destructive evaluation of growth during acclimatisation. This report describes the use of semi-automated and automated image analysis to quantify canopy growth of pineapple plantlets, during five months of acclimatisation. The canopy area progressively increased during acclimatisation, particularly after 90 days. Regression analyses were performed to determine the relationships between the automated image analysis and morphological indicators of growth. The mathematical relationships between estimations of the canopy area and the fresh and dry weights of intact plantlets, middle-aged leaves (D leaves) and roots showed determination coefficients (R2) between 0.84 and 0.92. We propose an appropriate tool for the simple, objective and non-destructive evaluation of pineapple plantlets growth, which can be generally applied for plant phenotyping, to reduce costs and develop streamlined pipelines for the assessment of plant growth.