Currently, with an increased requirement for new therapeutic strategies, preclinical drug testing or screening platforms have rapidly evolved in recent years. In comparison to traditional 2D cell cultures, 3D organoids or spheroids with or without scaffolds improve the microenvironment of in vitro cultures, advancing the in vitro biological observation and enabling mechanistic studies of drug reactions in the human tissue-like environment. 3D organoids and spheroids are straightforward to produce, and relatively uniform in size and shape. This helps to facilitate high throughput screening requirements. Spheroids and organoids have been applied in anti-cancer drug testing, toxicity evaluations, as well as mechanism studies for variable organ systems, including the intestine, liver, pancreas, brain, and heart. Among 3D cultures of spheroids and organoids, ‘tumour spheroids’ formed by dissociated tumour tissues or cancer cell lines are relatively simple in composition and commonly applied to anticancer drug screening. The ‘healthy organoids’ differentiated from hiPSCs/hESCs are more complex in cell composition, distribution, structure and function with higher similarity to in vivo organs, and have found applications in toxicity tests, personalised medicine, and therapeutic and mechanistic studies. In most cases, the multicellular 3D organoids are more resistant and stable in reaction to stimulations or chemicals in vitro , suggesting more accurate modelling of in vivo responses. Here, we review recent progress in human-origin organoid/spheroid systems and their applications in preclinical studies.