Genomics-based precision medicine has revolutionized oncology but also has inherent limitations. Functional precision oncology is emerging as a complementary approach that aims to bridge the gap between genotype and phenotype by modelling individual tumours in vitro. These patient-derived ex vivo models largely preserve several tumour characteristics that are not captured by genomics approaches and enable the functional dissection of tumour vulnerabilities in a personalized manner. In this Review, we discuss several examples of personalized functional assays involving tumour organoids, spheroids and explants and their potential to predict treatment responses and drug-induced toxicities in individual patients. These developments have opened exciting new avenues for precision oncology, with the potential for successful clinical applications in contexts in which genomic data alone are not informative. To implement these assays into clinical practice, we outline four key barriers that need to be overcome: assay success rates, turnaround times, the need for standardized conditions and the definition of in vitro responders. Furthermore, we discuss novel technological advances such as microfluidics that might reduce sample requirements, assay times and labour intensity and thereby enable functional precision oncology to be implemented in routine clinical practice.