Hypoxia stands in the pathophysiological background of some of the most common human diseases, such as heart attack and stroke. Since in addition to neurons, astrocytes also play a number of roles in the normal functioning of the nervous system, in this paper is investigated the expression of two intermediate filamentous cytoskeletal proteins of astrocytes: nestin, which is considered a marker of neural stem cells and glial fibrillar acidic protein (GFAP) which is a marker of mature astrocytes. Expression was investigated under conditions of normoxia (21% oxygen) and hypoxia (1% oxygen). In addition, the aim of this study was to investigate the application of two programs for semi-automatic signal analysis, Cell profiler and Fiji and to compare the results of quantification of the signal obtained by immunohistochemistry. After 8 days of differentiation from nerve stem cells, mature astrocytes were obtained. Half of these cells were transferred to an incubator with 1% oxygen for 24 hours while another part of the cells remained in the incubator at ambient oxygen concentration as a control. The immunohistochemical method revealed that the expression of nestin peaked at the beginning of differentiation and then gradually decreased. Hypoxia influenced expression of nestin and it was reduced. At the same time, the expression of GFAP increased in parallel to astrocyte differentiation. Hypoxia did not affect GFAP expression. Comparison of the results of immunohistochemical signal measurements obtained by the computer programs Cell profiler and Fiji showed that both programs give almost identical levels of statistical differences between the compared groups.