The microtubule (MT) cytoskeleton forms complex macromolecular assemblies with a plethora of MT-associated proteins (MAPs) that play fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates MT behaviour is an important step in understanding the physiological roles of various MT assemblies. To characterise how the ever-increasing number of MAPs control MT properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of MT-MAP assemblies. Analysing about fifty bona-fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique MT behaviours, such as MT coiling or hook formation, or liquid-liquid phase separation along MT lattice that initiates MT branching. We have thus established a powerful tool for a thorough characterisation of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.