MRI-targeted prostate biopsy improves detection of clinically significant prostate cancer (PCa). However, up to 70% of PCa lesions display intralesional tumor heterogeneity. Current target sampling strategies do not yet adequately account for this finding. This prospective study included 118 patients who underwent transperineal robotic assisted biopsy of the prostate. We identified a total of 58 PCa-positive PI-RADS lesions. We compared diagnostic accuracy of a target-saturation biopsy strategy to accuracy of single, two, or three randomly selected targeted biopsy cores and analysed potential clinical implications. Intralesional detection of clinically significant cancer (ISUP ≥ 2) was 78.3% for target-saturation biopsy and 39.1%, 52.2%, and 67.4% for one, two, and three targeted cores, respectively. Target-saturation biopsies led to a more accurate characterization of PCa in terms of Gleason score and reduced rates of significant cancer missed. Compared to one, two, and three targeted biopsy cores, target-saturation biopsies led to intensified staging procedures in 21.7%, 10.9, and 8.7% of patients, and ultimately to a potential change in therapy in 39.1%, 26.1%, and 10.9% of patients. This work presents the concept of robotic-assisted target saturation biopsy. This technique has the potential to improve diagnostic accuracy and thus individual staging procedures and treatment decisions.