For the first time, isoniazid (INH) bitterness value, threshold, and sensitivity (low, moderate, high, and extremely high) was determined in six human volunteers. INH demonstrated a large range in bitterness sensitivity. The current work demonstrates the design of a taste-masked isoniazid (INH)-loaded chitosan microspheres (INH-LCM) using an ionic-gelation and spray drying technique. A 2 full factorial design with three center points was employed to optimize and study the independent variables (chitosan concentration, sodium tripolyphosphate (TPP)-volume, feed rate, and air inlet temperature) effects on the critical quality attributes (percent yield [PY] and entrapment efficiency [EE]). Statistically significant models were developed for PY (p = 0.0357; adjusted R = 0.6078) and EE (p = 0.0190; adjusted R = 0.6713). A multicriteria prediction profiler was utilized to determine the optimum formulation and process parameters. Two verification batches confirmed excellent predictability and lot-to-lot consistency. In vitro dissolution was used to evaluate the taste masking ability of INH-LCM. The in vitro dissolution test of the optimized INH-LCM suggested that taste masking would be accomplished for the "low" and "moderate" bitterness taste sensitivity groups. Further in vitro and human volunteer taste panel studies with INH-LCM are required for better understand the potential taste masking capability for the "high" and "extremely high" bitterness taste sensitivity groups. The in vitro dissolution method and FTIR data analysis support that TPP crosslinked chitosan may provide taste masking by two mechanisms: (1) acts as a physical barrier and delays INH dissolution; and (2) provides a chemical barrier by forming hydrogen bonds between INH's bitter tasting amino group and chitosan.