By using a Laser Scanning Confocal Microscope combined with a High Temperature Furnace (HT-LSCM) for the in situ investigation of acicular ferrite (AF) formation in HSLA steels, new information about the mechanism of formation of this high toughness phase can be gained. Due to the utilization of an inert furnace atmosphere, the ability to accurately adjustment the austenitizing temperature and the well-controllable cooling conditions, the interactions between steel composition, austenite grain size, cooling rate and the fraction of AF formed have been analyzed in detail. The present work focuses on necessary adaptions and appropriate settings of the HT-LSCM for the investigation of the formation of AF. An intensive study of setting parameters for the visualization of the AF formation is done. Special attention is paid to the techniques by which austenite grain size is determined and the effect of austenite grain size on the fraction of AF formed. In addition, the complexity of studying manganese alloyed steels in the HT-LSCM has been elucidated.