The delivery of genetic material (DNA and RNA) to cells can cure a wide range of diseases but is limited by the delivery efficiency of the carrier system. Poly β-amino esters (pBAEs) are promising polymer-based vectors that form polyplexes with negatively charged oligonucleotides, enabling cell membrane uptake and gene delivery. pBAE backbone polymer chemistry, as well as terminal oligopeptide modifications, define cellular uptake and transfection efficiency in a given cell line, along with nanoparticle size and polydispersity. Moreover, uptake and transfection efficiency of a given polyplex formulation also vary from cell type to cell type. Therefore, finding the optimal formulation leading to high uptake in a new cell line is dictated by trial and error, and requires time and resources. Machine learning (ML) is an ideal in silico screening tool to learn the non-linearities of complex data sets, like the one presented herein, with the aim of predicting cellular internalisation of pBAE polyplexes. A library of pBAE nanoparticles was fabricated and the uptake studied in 4 different cell lines, on which various ML models were successfully trained. The best performing models were found to be gradient-boosted trees and neural networks. The gradient-boosted trees model was then analysed using SHapley Additive exPlanations, to interpret the model and gain an understanding into the important features and their impact on the predicted outcome.